Essential role of extracellular charged residues of the human CCK(1) receptor for interactions with SR 146131, SR 27897 and CCK-8S

We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.     

The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK(1) receptor

1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK₁ receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK₁ receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [³H]SR 27897 binding properties, Lys¹¹⁵, Lys¹⁸⁷, Phe¹⁹⁸, Trp²⁰⁹, Leu²¹⁴ and Asn³³³. In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe⁹⁷, Gly¹²², Phe¹⁹⁸, Trp²⁰⁹, Ile²²⁹, Asn³³³, Arg³³⁶ and Leu³⁵⁶ or increased it, Tyr⁴⁸, Cys⁹⁴, Asn⁹⁸, Leu²¹⁷ and Ser³⁵⁹. Only mutations of Phe¹⁹⁸, Trp²⁰⁹ and Asn³³³ affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK₁ receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.     

The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK1 receptor

1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK₁ receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK₁ receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [³H]SR 27897 binding properties, Lys¹¹⁵, Lys¹⁸⁷, Phe¹⁹⁸, Trp²⁰⁹, Leu²¹⁴ and Asn³³³. In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe⁹⁷, Gly¹²², Phe¹⁹⁸, Trp²⁰⁹, Ile²²⁹, Asn³³³, Arg³³⁶ and Leu³⁵⁶ or increased it, Tyr⁴⁸, Cys⁹⁴, Asn⁹⁸, Leu²¹⁷ and Ser³⁵⁹. Only mutations of Phe¹⁹⁸, Trp²⁰⁹ and Asn³³³ affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK₁ receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.     

Characterisation of the effects of SR146131, a novel non-peptide CCK(1) receptor agonist, on IMR-32 human neuroblastoma cells

The effect of ?2-[4-(4-chloro-2, 5-dimethoxy-phenyl)-5-[2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoy l]-5, 7-dimethyl-indol-1-yl?-acetic acid (SR146131), a novel non-peptide agonist of cholecystokinin (CCK) CCK(1) receptors, was compared to the effect of sulphated cholecystokinin octapeptide (CCK-8-S) on CCK(1) receptors of the human neuroblastoma cell line IMR-32. SR146131 inhibited [125I]CCK-8-S binding to IMR-32 cells at nanomolar concentrations. SR146131 and CCK-8-S increased intracellular free Ca(2+) levels ([Ca(2+)](i)) in the same concentration range (EC(50)=6+/-2.3 and 1.3+/-0.14 nM, respectively). Although the shape of the [Ca(2+)](i) increase induced by CCK-8-S and SR146131 was slightly different, extracellular Ca(2+) removal affected the response of both compounds to a similar degree, and the response of both compounds was essentially due to Ca(2+) release from intracellular stores. This was also confirmed by measuring the [Ca(2+)](i) response of single cells: both compounds induced [Ca(2+)](i) oscillations at subnanomolar concentrations and elicited a large peak increase in [Ca(2+)](i) at higher concentrations (EC(50)=0.5+/-0.04 and 5.7+/-1.9 nM for CCK-8-S and SR146131, respectively). Both CCK-8-S and SR146131 induced a sustained increase of phosphoinositide turnover in these cells, and acted at similar concentrations (EC(50)=2.7+/-0.7 and 6+/-3.1 nM, respectively), although the maximal effect of SR146131 was somewhat lower than the effect of CCK-8-S. These data show that SR146131 activates human CCK(1) receptors on IMR-32 cells in a manner and with a potency similar to that of CCK-8-S.     

SR146131, a cholecystokinin-A receptor agonist,antagonizes prepulse inhibition deficits produced by dizocilpine and DOI

Abstract Rationale. Converging evidence has demonstrated that cholecystokinin (CCK) inhibits mesolimbic brain dopamine (DA) function via activation of CCK-A (CCK-1) receptors. These effects of CCK have stimulated interest in the potential use of CCK agonists as antipsychotic drugs. Most research on the antipsychotic-like drug effects of CCK has used CCK or CCK analogues that nonselectively activate both CCK-A and CCK-B (CCK-2) receptors, which may produce opposite effects. SR146131, a CCK-A selective nonpeptide agonist, has recently been developed (Sanofi-Synthelabo). Objective. To determine whether SR146131 exhibits antipsychotic-like qualities in the prepulse inhibition (PPI) paradigm. Methods. We performed experiments to determine whether SR146131 (vehicle, 0.01, 0.1, 1.0 mg/kg) would attenuate PPI deficits induced by amphetamine (2.0 mg/kg), an indirect dopamine agonist, and dizocilpine (0.1 mg/kg), a noncompetitive N-methyl-D-aspartate (NMDA) antagonist. Since SR146131 demonstrated significant effects on PPI disrupted by the noncompetitive NMDA antagonist, an effect associated with drugs that inhibit serotonin (5HT)2A transmission, we also tested the effects of SR146131 on PPI disruption produced by 2,5-dimethoxy-4-iodoamphetamine (DOI, 0.5 mg/kg), a direct 5HT2A agonist. Results. SR146131 did not significantly affect startle magnitude, baseline PPI, or amphetamine-induced PPI deficits. However, it dose-dependently antagonized dizocilpine and DOI-induced PPI deficits. Conclusions. The lack of an effect of SR146131 on amphetamine-induced disruption of PPI suggests that a selective nonpeptide CCK-A agonist may not produce antipsychotic-like effects on dopamine transmission. However, the unexpected effects of SR146131 on dizocilpine and DOI-induced PPI deficits are consistent with the effects of drugs that inhibit transmission in the 5HT2A receptor system, including atypical antipsychotic drugs. Possible mechanisms underlying these findings are discussed. Electronic Publication     

Pharmacological evidence for putative CCK(1) receptor heterogeneity in human colon smooth muscle

1. The pharmacology of the cholecystokinin CCK(1) receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. 2. Saturation analysis of the interaction between the radiolabelled, selective CCK(1)-receptor antagonist, [(3)H]-L-364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. 3. Competition studies, using a range of structurally diverse, CCK-receptor selective ligands provided further evidence for CCK(1) receptor heterogeneity in human colon tissue (n(H) values significantly less than unity for SR27897=0.77+/-0.07, 2-NAP=0.73+/-0.03, YM220=0.70+/-0.09 and PD-134,308=0.83+/-0.01). Moreover, the competition data for SR27897, 2-NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. 4. The data obtained are consistent with the presence of a single CCK(1) receptor binding site in the gallbladder but not in the colon. A two-site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established.     

Neurobehavioural effects of SR 27897, a selective cholecystokinin type A (CCK-A) receptor antagonist

Summary The activity of SR 27897, a potent and selective CCK-A vs CCK-B receptor antagonist (Ki = 0.2 nM on guinea-pig pancreas vs 2000 nM on rat brain) was studied on behavioural, electrophysiological and biochemical effects induced by peripheral or central injection of CCK-8S. For comparative purposes, devazepide, a reference CCK-A receptor antagonist, was investigated in these same models. CCK-induced hypophagia and CCK-induced hypolocomotion in rats, two behavioural changes associated with the stimulation of peripheral CCK-A receptors, were dose-dependently antagonized by SR 27897 (ED50 = 0.003 and 0.002 mg/kg i.p., respectively) and devazepide (ED50 = 0.02 and 0.1 mg/kg i.p., respectively). CCK-induced decrease of cerebellar cGMP levels in mice was also reduced by SR 27897 (ED50 = 0.013 mg/kg) and by devazepide (0.084 mg/kg). The CCK-induced turning behaviour after intrastriatal injection in mice, and the potentiation of the rate suppressant activity of apomorphine on rat DA neurons, were blocked by higher doses of SR 27897 and devazepide, consistent with the probable central origin of these effects. The respective ED50s were 0.2 mg/kg i.p. for SR 27897 and 4.9 mg/kg i.p. for devazepide in the former model, while the respective minimal effective doses were 1.25 and 5 mg/kg i.p. in the latter test. In most tests the i.p./p.o. ratio for SR 27897 was near unity, suggesting a high oral bioavailability of the compound. Taken together, these findings support the notion that SR 27897 behaves as a potent CCK-A antagonist able to cross the blood brain barrier.Correspondence to P. Soubrié at the above address     

Vasopressin V2 (SR121463A) and V1a (SR49059) receptor antagonists both inhibit desmopressin vasorelaxing activity

Although [Arg(8)]vasopressin is a potent vasoconstrictor, it possesses vasorelaxant properties manifested either after vasopressin V1 receptor blockade or directly in some vascular beds. The nature of the receptor involved in the vasorelaxant effect of [deamino-Cys(1) D-Arg(8)]vasopressin (desmopressin), a vasopressin V2 receptor agonist, was studied on rat precontracted aortic rings by the use of highly selective new non-peptide vasopressin receptor antagonists. The present study demonstrates for the first time that desmopressin relaxant effect is antagonized by the vasopressin V2 receptor antagonist SR121463A, but also by the vasopressin V1A receptor antagonist SR49059, suggesting that desmopressin-induced relaxation is mediated by a receptor subtype sharing both V1A and V2 pharmacological profiles.     

Opposite modulation of apomorphine- or amphetamine-induced stereotypy by antagonists of CCK receptors

Stereotyped behavior is elicited by activation of dopaminergic systems with drugs such as apomorphine and amphetamine. In previous studies, we have reported that the sulfated cholecystokinin octapeptide (CCK-8) decreased apomorphine-induced stereotypy in animals with normal and supersensitive dopamine receptors. The aim of the present study was to evaluate the effects of CCK(1) and CCK(2) receptor antagonists on stereotyped behavior induced by apomorphine or amphetamine. Rats were pretreated with the CCK(1) (SR 27897B; 1-[[2-(4-(2-chlorophenyl) thiazol-2-yl) aminocarbonyl]indolyl]acetic acid; 500 microg/kg; i.p.) or CCK(2) (L-365,260; 3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5 phenyl-1H-1, 4-benzodiazepine-3-yl)-N'-(3-methyl phenyl)-urea; 500 microg/kg; i.p. ) receptor antagonists or saline 15 min before apomorphine (0.6 mg/kg; s.c.) or amphetamine (9.0 mg/kg; i.p.) injection. Both CCK(1) and CCK(2) receptor antagonists significantly increased apomorphine-induced stereotypy. In contrast, only the blockade of CCK(2) receptors significantly decreased amphetamine-induced stereotypy. The results suggest a dual opposite mechanism for CCK-dopamine interactions. These data also suggest that both apomorphine- and amphetamine-induced stereotypy should be used whenever effects of drugs acting on dopaminergic systems are being assessed.     

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.     

Functional comparisons of gastrin/cholecystokinin receptors in isolated preparations of gastric mucosa and ileum.

1. The gastrin cholecystokinin (CCK) receptors mediating stimulation of acid secretion in rat isolated gastric mucosa (RGM) and contraction in guinea-pig isolated ileum longitudinal muscle-myenteric plexus (GPI) have been characterized by use of peptide agonists and the non-peptide antagonists, lorglumide, devazepide and L-365,260. 2. In RGM, gastrin peptides (sulphated gastrin heptadecapeptide (G-17), non-sulphated (ns) G-17 and pentagastrin) were potent agonists of acid secretion (EC50 values of 4.3, 16 and 27 nM respectively). Sulphated CCK octapeptide (CCK-8) was also a potent agonist, (EC50 = 0.9 nM), but was less efficacious, producing a lower maximal response. In contrast, in GPI, CCK-8 was a potent full agonist (EC50 = 1.4 nM) and was more than 1000 times more potent than the gastrin peptides in producing a sustained contractile response. 3. In GPI, CCK-8 (0.1 to 100 nM) produced sustained contractile responses, whilst CCK-4 (3 to 1000 nM) produced transient responses. These responses had different sensitivities to atropine (1 microM), suggesting that more than one receptor may mediate contraction in this tissue. 4. In RGM, L-365,260 was the most potent antagonist of pentagastrin-stimulated acid secretion (pA2 = 7.6). This functional affinity estimate was similar to that for L-365,260 as an antagonist of excitatory responses in rat ventromedial hypothalamic slices (Kemp et al., 1989) but differed from binding affinity estimates in guinea-pig cortex and gastric glands (Freidinger, 1989). 5. In GPI, devazepide, L-365,260 and lorglumide yielded different affinity estimates when compared against CCK-8 and CCK-4 or pentagastrin respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A-71623, a selective CCK-A receptor agonist, suppresses food intake in the mouse, dog, and monkey.

The anorectic actions of cholecystokinin (CCK)-8 and of a selective CCK-A agonist, A-71623, were examined in CD1 mice, beagle dogs, and cynomolgus monkeys. A-71623 suppressed intakes in all species tested, and the effects were blocked by a selective CCK-A antagonist, A-70104. In the dog only, CCK-8 was more potent on a molar basis compared to A-71623, although the effects of both CCK agonists were more short-lived in the dog compared to the other species tested. Our results support other evidence suggesting that the anorectic actions of exogenous application of CCK-8 in these species are mediated via stimulation of the CCK-A receptor subtype.     

A novel effect of rebamipide: generation of [Ca(2+)](i) oscillations through activation of CCK(1) receptors in rat pancreatic acinar cells

The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.     

Characterization of CCK receptors in a novel smooth muscle preparation from the guinea-pig stomach by use of the selective antagonists CI-988, L-365,260 and devazepide.

1. The cholecystokinin receptors mediating motor responses in a novel smooth muscle preparation from the corpus region of the guinea-pig stomach have been characterized by use of five agonist peptides and the antagonists CI-988, L-365,260 and devazepide. 2. Mucosa-denuded strips of circular muscle were contracted in a concentration-dependent manner by the five cholecystokinin (CCK)-related peptides CCK-8S, pentagastrin, gastrin-I, CCK-8US and CCK-4. 3. CI-988 was a powerful antagonist of the response to pentagastrin with an affinity (pKB = 9.49) similar to that obtained in CCKB receptor binding assays. With CCK-8S as the agonist, CI-988 was approximately 1000 fold less powerful as an antagonist. 4. Devazepide powerfully blocked responses to CCK-8S with an affinity (pKB = 9.54) that was in agreement with reported functional data obtained in pancreatic amylase secretion studies, a system exhibiting CCKA receptor activity. Devazepide displayed lower affinity against pentagastrin than against CCK-8S. 5. CI-988 blocked responses to pentagastrin in an insurmountable manner in the presence of 3 nM devazepide; a concentration previously shown to block the CCKA receptor. The nature of the antagonism observed with L-365,260 was unaltered by the presence of devazepide. 6. The guinea-pig stomach corpus smooth muscle preparation contains both subtypes of CCK receptor and will be useful as a pharmacological tool for investigating the functional effects of novel CCK ligands.     

Pharmacology of a cholecystokinin receptor on 5-hydroxytryptamine neurones in the dorsal raphe of the rat brain.

1. The effect of bath application of sulphated cholecystokinin octapeptide (CCK-8) was studied on neurones in slices containing rat raphe nucleus. 2. Intracellular recordings were made from neurones in the dorsal raphe nucleus. Some of the neurones with the characteristics of 5-hydroxytryptamine (5-HT)-containing cells which were inhibited by 5-HT and excited by noradrenaline were excited by cholecystokinin. The response to cholecystokinin was dose-dependent over the range 10 to 1000 nM. 3. The response to CCK-8 persisted in the presence of tetrodotoxin. Either reduction of extracellular calcium or addition of 25 mM magnesium did not block the CCK response, suggesting it was mediated by receptors located on the membrane of the raphe neurones. 4. The agonist and antagonist specificity of the CCK response was determined. The CCKB selective agonist, pentagastrin, was inactive when applied at concentrations up to 10 microM. the CCKA receptor antagonist L-364,718 (1 to 100 nM) blocked the response to cholecystokinin. Much higher (1-10 microM) concentrations of the CCKB receptor antagonist L-365,260 were required for inhibition of the CCK response. 5. These data support the existence of a CCK receptor, located on raphe neurones in the rat, with a pharmacological profile very similar to that described for the CCKA type.     

The cholecystokinin analogues JMV-180 and CCK-8 stimulate phospholipase C through the same binding site of CCK(A) receptor in rat pancreatic acini

1. This study was designed to address the controversy related to the involvement of phospholipase C in the signalling pathway linked to CCK(A) receptor stimulation by the cholecystokinin analogue JMV-180, a full agonist for amylase release, in rat pancreatic acini. 2. JMV-180 was shown to stimulate phospholipase C by measuring the incorporation of [(32)P]-orthophosphoric acid ([(32)P]-Pi) into phosphatidic acid (PtdOH) and phosphatidylinositol (PtdIns). Both responses elicited by JMV-180 were time and concentration dependent. Maximal effects elicited by JMV-180 were 39.08+/-0.72 and 8.02+/-0.40% for the labelling of [(32)P]-PtdIns and [(32)P]-PtdOH, respectively, as compared to the maximal effects of CCK-8, a full agonist of the CCK(A) receptor. 3. [(32)P]-Pi incorporation into PtdOH and PtdIns was sensitive to lithium, demonstrating that both responses are a consequence of phospholipase C activation. However, since lithium blocks the phosphoinositide cycle by an uncompetitive mechanism, its effect was only apparent at high concentrations of CCK-8 (>10 pM), which elicited stimuli above 20 and 60% of the maximal [(32)P]-PtdOH and [(32)P]-PtdIns labelling, respectively. 4. JMV-180 inhibited the incorporation of [(32)P]-Pi into PtdOH and PtdIns as stimulated by CCK-8, down to its own maximal effect. The estimated IC(50) values for the inhibition curves were not significantly different from those calculated assuming the same single binding site for both agonists. These results indicated that the well established role of JMV-180 as a partial agonist for CCK(A) receptor-linked signalling responses, also applies for the stimulation of phospholipase C. 5. The comparison of CCK-8 and JMV-180 dose-response curves of amylase release to those of PtdIns and PtdOH labelling with [(32)P]-Pi showed the existence of an amplification mechanism between phospholipase C and amylase release for both agonists. 6. In conclusion, we show that JMV-180, as well as CCK-8, stimulate phospholipase C upon interaction with the same binding site at the CCK(A) receptor in rat pancreatic acini.     

A cholecystokinin-1 receptor agonist (CCK-8) mediates increased permeability of brain barriers to leptin

BACKGROUND AND PURPOSE: Leptin regulates energy expenditure and body weight by acting both on the hypothalamus and on peripheral targets. Central actions of leptin are enhanced by cholecystokinin (CCK). The interaction between leptin and CCK makes physiological sense, as rats lacking CCK1 receptors are resistant to peripheral leptin but not to leptin directly infused into the brain. We have recently reported that CCK enhances leptin effects by increasing the entry of leptin into the CNS. The aim of this work was to further characterize the effect of CCK (10 microg kg(-1)) on leptin kinetics as well as the CCK receptor subtype involved in the interaction between CCK and leptin. EXPERIMENTAL APPROACH: Experiments were carried out both in free-feeding and in fasted rats receiving a single dose of leptin (100 microg kg(-1); i.p.). Parameters analysed over the next 6 h were plasma and cerebrospinal fluid concentrations of leptin. KEY RESULTS: We observed that CCK-8 depressed the increase in plasma leptin that followed the i.p. injection and simultaneously increased leptin concentration in the cerebrospinal fluid from 92+/-25 to 230+/-24 pg mL(-1) (P<0.05). The effect of CCK-8 was totally prevented by the CCK1 receptor antagonist, SR-27,897 (0.3 mg kg(-1), s.c.), but not by the CCK2 receptor antagonist, L-365,260 (1 mg kg(-1)). CONCLUSIONS AND IMPLICATIONS: These results show that CCK plays a role in regulating the access of leptin to the brain and suggest that CCK analogues, acting on CCK1 receptors, might be useful drugs in improving leptin actions within the brain.     

Pharmacological characterization of rebamipide: its cholecystokinin CCK1 receptor binding profile and effects on Ca2+ mobilization and amylase release in rat pancreatic acinar cells

We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 microM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+ -mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.     

Inhibition of the hypothalamic-pituitary-adrenal axis in food-deprived rats by a CCK-A receptor antagonist

The circadian activity of the hypothalamic-pituitary-adrenal (HPA) axis is regulated by caloric flow in rats. During the dark cycle, it has been shown that, in fasted rats, the time-course profile of plasma concentrations of adrenocorticotropin (ACTH) and corticosterone parallels the profile of food intake in ad libitum fed animals. Cholecystokinin (CCK) is involved in regulating food intake in rodents. CCK-8 reduces food intake by acting on CCK-A receptors subtype. This work aims at establishing an eventual relationship between the modulatory role of CCK on food intake and its effect on HPA axis activity during fasting. We studied the effect of CCK-A and CCK-B receptor antagonists on food intake during the first period of the dark cycle. Under these conditions we observed that the CCK-A receptor antagonist, SR-27897 (0.3 mg kg(-1)), but not the CCK-B receptor antagonist, L-365260 (1 mg kg(-1)), increases food-intake. In a second series of experiments we observed that the increase of both ACTH and corticosterone plasma level elicited by fasting, was prevented by SR-27897, but not by L-365260. These results indicate that CCK-A receptor blockade during fasting prevents the activation of the HPA axis.     

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.