The cholecystokinin type A receptor (CCKAR) gene has been found to be associated with positive symptoms in patients with schizophrenia but the results reported to date are inconsistent. Considering the involvement of allelic heterogeneity in poor replication of the CCKAR finding, we genotyped five single nucleotide polymorphisms (SNPs) located in the 5' putative regulatory region of the CCKAR gene in a Chinese case-control sample and then applied the 5-SNP haplotype analysis to extract allelic heterogeneity information. The results showed that three individual haplotypes were strongly associated with increased risk of schizophrenia (corrected P = 2.9 × 10(-4), P = 2.5 × 10(-5), and P = 1.4 × 10(-5), respectively) and their combination gave an odds ratio (OR) of 6.12 with 95% CI 3.67-10.21 (P = 6.7 × 10(-15)). The haplotypes were also associated with some clinical symptoms including hallucination, suspiciousness, and hostility. Our work provided further evidence in support of the CCKAR hypothesis of schizophrenia and also suggested that haplotype-based association analysis may be a powerful approach for identification of allelic heterogeneity of a disease-underlying gene, which is very likely to be attributable to poor replication of an initial finding due to the reduction of sample power and the complexity of genetic architectures.

Alpha lipoic acid (α -LA) is a naturally occurring antioxidant and metabolic enzyme co-factor. Recently, α -LA has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of α -LA on non-small cell lung cancer (NSCLC) development remain unclear.

The CCK-8 assay was used to assess cell proliferation in NSCLC cell lines after α -LA treatment. The expression of growth factor receptor-bound protein 2 (Grb2), cyclin-dependent kinase (CDK)-2, CDK4, CDK6, Cyclin D3, Cyclin E1, Ras, c-Raf, epidermal growth factor receptor (EGFR), ERK1/2 and activated EGFR and ERK1/2 was evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in NSCLC cells via specific siRNA-mediated knockdown.

α -LA dramatically decreased NSCLC cell proliferation by downregulating Grb2; in contrast, Grb2 overexpression significantly prevented α-LA-induced decrease in cell growth in vitro. Western blot analysis indicated that α-LA decreased the levels of phospho-EGFR, CDK2/4/6, Cyclins D3 and E1, which are associated with the inhibition of G1/S-phase transition. Additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. In addition, α-LA exerted greater inhibitory effects than gefitinib on NSCLC cells by preventing EGF-induced EGFR activation.

For the first time, these findings provide the first evidence that α-LA inhibits cell proliferation through Grb2 by suppressing EGFR phosphorylation and that MAPK/ERK is involved in this pathway.

Rats fed raw soy flour (RSF) show pancreatic growth due to excessive cholecystokinin (CCK) release. Soybean trypsin inhibitors are implicated, but rats fed soybean lectin also showed pancreatic growth. Therefore, we studied the effect of soybean lectin on pancreatic protein secretion in anesthetized rats. Intraduodenal administration of 30 mg of RSF stimulated a 1-h integrated rise in pancreatic protein output of 2.2 +/- 1.1 mg/h (mean +/- SE) in rats with bile pancreatic (BP) juice returned to the duodenum. Selective removal of the lectin by affinity to N-acetyl-D-galactosamineagarose abolished the response (-0.1 +/- 0.2 mg/h). Adding back the 84 micrograms of lectin restored the output of 2.2 +/- 0.9 mg/h. With BP juice returned to the duodenum, 84 micrograms of lectin required the added presence of protein and protease inhibitors to have this effect. However, when BP juice was not returned, 84 micrograms of lectin given alone produced a pancreatic response of 3.2 +/- 1.3 mg/h. Plasma CCK concentrations rose significantly from 6.6 +/- 1.9 to 14.3 +/- 2.9 pmol/l, and the pancreatic response was abolished by CCK-A receptor blockade (0.0 +/- 0.1 mg/h). We conclude that soybean lectin plays a major role in the acute stimulation of pancreatic protein secretion by RSF. The lectin releases CCK and the effect is mediated by CCK-A receptors.

Latent inhibition (LI) is a behavioural paradigm in which repeated exposure to a stimulus without consequence inhibits the formation of any new associations with that stimulus. To the extent that LI reflects a process of leaming to ignore irrelevant stimuli, disrupted LI has been suggested as an animal model for the attentional deficits observed in schizophrenia. The antipsychotic potential of cholecystokinin (CCK) stems from its colocalization with dopamine (DA) in the mesolimbic pathway, where it demonstrates both excitatory and inhibitory effects on dopaminergic activity. This may be explained by mediation through different receptor subtypes. A variety of hypotheses has emerged regarding the potential clinical application of subtype-selective CCK-based drugs. The present experiments examined the effects on LI of two selective CCK(A) ligands: PD-140,548 (a CCK(A) antagonist, Experiment 1: 0.001, 0.01, and 0.1 mg/kg) and A-71623 (a CCK(A) agonist, Experiment 2: 0.02, 0.05, and 0.1 mg/kg). In both experiments, the effects of haloperidol (0.1 mg/kg) were also investigated. Animals receiving 0.1 mg/kg of haloperidol or 0.001 or 0.1 mg/kg (but not 0.01 mg/kg) of PD-140,548 treated the preexposed stimulus as irrelevant after a low number of preexposures. In contrast, no facilitatory effect on LI was detectable at any of the A-71623 doses. The finding that A-71623 failed to enhance LI indicates that it is unlikely that this compound would have any antipsychotic effect within the clinical setting. Considering the facilitatory effect exerted by PD-140,548 on LI, it is probable that the inhibition of CCK activity might prove a more promising strategy for the pharmacological treatment of schizophrenia.

Cholecystokinin (CCK), a neuropeptide which fulfills almost all criteria for neurotransmitter status, has been co-localized with dopamine in midbrain mesolimbic and mesocortical neurons that have been implicated in the pathogenesis of schizophrenia. Preclinical research suggests that CCK may in part act to enhance central dopaminergic activity. In an attempt to evaluate the role of CCK relative to the dopamine hyperactivity hypothesis of schizophrenia, in the present investigation the putative CCK receptor antagonist, proglumide, was administered to four schizophrenic patients in a double-blind, placebo-controlled study. All patients were receiving concurrent neuroleptic medication, but were still significantly symptomatic. Proglumide was without effect on the patients' psychosis ratings. Potential reasons for this negative finding are discussed.

BACKGROUND

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.

Dipsacus saponin C (DSC) administered intrathecally (i.t.) showed antinociceptive effect in a dose-dependent (from 3.75 to 30 microg) manner as measured by the tail-flick assay. The antinociception induced by DSC at the dose of 30 microg reached at peak 7.5 min and almost returned to the control level after 60 min. 5-Amino-valeric acid (5-AVA, a GABA(A) receptor antagonist, from 1 to 20 microg) and SR 95531 (a GABA(B) receptor antagonist, from 0.1 to 2 ng) dose-dependently attenuated i.t. administered DSC-induced increase of the inhibition of the tail-flick response. The i.t. injection of yohimbine (an alpha(2)-adrenergic receptor antagonist, from 1 to 20 microg) and methysergide (a serotonin receptor antagonist, from 1 to 20 microg), but not naloxone (from 2 to 8 microg), significantly attenuated inhibition of the tail-flick response induced by DSC (30 microg) administered i.t. Sulfated cholecystokinin (CCK, from 0.05 to 0.5 ng) injected i.t. significantly reduced the inhibition of the tail-flick response induced by DSC (30 microg) administered i.t. Our results suggest that DSC shows an antinociceptive effect when it is administered spinally and GABA(A), GABA(B), alpha(2)-adrenergic and serotonin receptors located at the spinal cord level, but not opioid receptors, may be involved in DSC-induced antinociception. Furthermore, CCK may play an important role for the modulation of i. t. injected DSC-induced antinociception.

1. The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[N alpha-tert-butoxicarbonyl)L-tryptophyl] amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158. 2. IQM-95,333 displaced [3H]-CCK-8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50>> 10 microM). IQM-95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. 3. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCKA receptors. 4. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCKA receptor-mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. 5. Low doses (50-100 micrograms kg-1, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCKA receptors. 6. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10-5,000 micrograms kg-1). Typical CCKA and CCKB antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range. 7. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. 8. In conclusion, IQM-95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic-like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

The effects of cholecystokinin (CCK) receptor ligands were studied in the rat safety signal withdrawal conflict procedure, an operant paradigm sensitive to both anxiolytic and anxiogenic compounds. In this procedure, behavioural suppression of lever pressing for food was induced by the withdrawal of a conditioned signal for safety without the usual presentation of a conditioned signal for danger. The compounds tested were selective CCK-B antagonists [CI-988 (0.01-1 mg/kg SC), L-365,260 (0.004-2 mg/kg IP) and LY 262,691 (0.001-1 mg/kg SC)], CCK-B agonists [CCK-4 (0.01-1 mg/kg SC) and BC 264 (0.004-1 mg/kg IP)] and CCK-A antagonists [devazepide (0.001-1 mg/kg SC) and lorglumide (0.01-1 mg/kg SC)]. None of these drugs induced the expected behavioural effects, i.e. an anxiolytic-like release of the behavioural suppression with CCK-B and, possibly, CCK-A antagonists and/or a further reduction of lever pressing with CCK-B agonists, indicative of an anxiogenic-like potential. In contrast, the established anxiolytic lorazepam (0.06-0.25 mg/kg IP), as well as diazepam (2 mg/kg IP) and buspirone (0.25 mg/kg SC) used as positive control drugs, released the suppression of pressing for food during the period associated with the safety signal withdrawal, whereas picrotoxin (1 mg/kg IP), used as an anxiogenic control, further reduced responding during this conflict period. The present results contrast with a series of published data suggesting the involvement of CCK processes in anxiety-related behaviour in rodent models such as the elevated plus-maze or the light:dark two compartment test, and in panic disorders in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin octapeptide (CCK-8) and the peptide analog ARL 14294, formerly FPL 14294, [Hpa(SO3H)-Met-Gly-Trp-Met-Asp-N(Me)Phe-NH2], have been reported to induce satiety by interaction with the CCK-A receptor subtype. ARL 15849 [Hpa(SO3H)-Nle-Gly-Trp-Nle-N(Me)-Asp-Phe-NH2] is an improved ARL 14294 analog with enhanced CCK-A receptor selectivity, greater stability, and a longer duration of action. The affinity of ARL 15849 for the CCK-A receptor (Ki = 0.034 nM) is 6,600 fold greater than for the CCK-B receptor (Ki = 224 nM), whereas CCK-8 and ARL 14294 are nonselective. Although comparable in potency to contract isolated gallbladder and induce pancreatic phosphatidylinositol hydrolysis, ARL 15849 is 3- and 100-fold more potent than ARL 14294 and CCK-8, respectively, to inhibit 3-h feeding in rats. The duration of feeding inhibition was significantly longer for ARL 15849 (>5 h), compared to equipotent doses of ARL 14294 (3 h), and CCK-8 (1 h). Intranasal administration of ARL 15849 inhibits feeding in beagle dogs with a greater separation between doses that induce emesis and those that inhibit feeding. Therefore, ARL 15849 is a potent, selective, intranasally active anorectic agent which may be useful in the treatment of eating disorders.

New (R)-4-benzamido-5-oxopentanoic acid derivatives were synthesized by a stereoconservative procedure and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK-B) CCK receptors, or the binding of [3H]pentagastrin to rabbit gastric glands, as well as to inhibit, in vivo, the acid secretion induced by pentagastrin infusion in the perfused rat stomach. The parent compound of this series (lorglumide) is the first nonpeptidic, potent and selective antagonist of the CCK-A receptor. Chemical manipulations of the structure of lorglumide led to the discovery of selective antagonists of the CCK-B/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives exhibit different affinities with rabbit gastric gland cells and rat cortex membranes, suggesting that the stomach gastrin receptor (arbitrarily termed CCK-B1 receptor) is not as closely related to the CCK central receptor (termed CCK-B2) as previously hypothesized. The antigastric activity of the most potent compound of the series, i.e. (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid (compound 28, CR 2194) was further evaluated in vivo: in the first hour after administration the compound inhibits acid secretion induced by pentagastrin infusion, in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch), with ID50s (mg/kg) of 15.5 (iv) (cat), 8.7 (IV) (dog) and 24.2 (oral) (Heidenhain dog). The characteristics of CR 2194, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure, and the activity after oral administration, indicate that this compound is a useful tool in the study of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use.

Cholecystokinin (CCK) causes relaxation of the cat lower esophageal sphincter (LES) by stimulating CCK receptors on the noncholinergic, nonadrenergic inhibitory neurons and causes contraction by stimulating CCK receptors on the sphincter muscle. Studies were performed in anesthetized cats to identify differences between the two CCK receptors by investigating the structure-activity relationships of various fragments of CCK or gastrin molecule. Lower esophageal sphincter pressures were monitored continuously, using continuously perfused catheters, and agents were administered intravenously or close intraarterially. Based on their doses and the presence or absence of tetrodotoxin pretreatment, CCK analogues produced either relaxation or contraction of the sphincter. The relative potencies of CCK analogues on the inhibitory (neural) response were CCK-8 greater than G-17-I greater than or equal to dCCK greater than CCK-4(1:1/14,000: 1/15,000: 1/335,000). Sulfated gastrin was nearly as potent as CCK-8. The relative potency of these agents on the contractile (muscle) response was CCK-8 = G-17-I greater than or equal to dCCK-8 greater than CCK-4 (1:1:1/4.5: 1/2000). Deamidated CCK-8 was inactive. Proglumide shifted the dose-response curves of the inhibitory as well as excitatory effects of CCK analogues to the right. These studies show that there are two distinct species of CCK receptors: (a) The CCK alpha receptors, present on the inhibitory neurons, are very discriminative and are critically dependent on SO4; and (b) the CCK beta receptors, present on the sphincter muscle, are not discriminative and are not critically dependent on SO4. Nonsulfated gastrin may share the CCK beta receptors with CCK.

Cholecystokinin (CCK), a gastrin-like neuropeptide, exists in the central nervous system in several forms. The octapeptide (CCK-8) occurs in predominantly sulfated form (CCK-8S), and the tetrapeptide (CCK-4) occurs in smaller but significant quantities. This review highlights recent developments in preclinical and clinical research into the potential role for CCK in mediating anxiety states. Relevant animal and human studies of administration of CCK agonists are discussed, as well as recent data regarding the concentration of CCK-8S in cerebrospinal fluid from patients with panic disorder, bulimia nervosa, and obsessive compulsive disorder. Finally, the development of agents that specifically antagonize CCK receptors will be described, as will potential therapeutic uses for these new compounds.

The synthesis and biological evaluation of a series of 2-substituted 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a kappa-opioid agonist and a cholecystokinin-A (CCK-A) antagonist, are reported. The radioligand binding models used in these studies were [(125)I](BH)-CCK-8 in rat pancreas (CCK-A), [(3)H]-(MENLE(28,31))-cck-8 in guinea pig cerebral cortex (CCK-B), and [(3)H]U-69593 (kappa(1)), [(3)H]DAMGO (mu), and [(3)H]DADLE (delta) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for kappa-opioid receptors. In particular, the 2-thienyl derivative 7A(X = H) with a K(i) = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig kappa (kappa(1))-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure-affinity relationships in this class of receptors.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a new model of a congenital defect of the CCK-A receptor gene and should be useful for determining CCK-A receptor function. Since the CCK-A receptor plays an important role in gastrointestinal function, in the present study we examined, using OLETF rats, the hypothesis that a defect of the CCK-A receptor may influence gastric mucosal integrity. The gastric mucosal integrity was evaluated by the severity of gastric mucosal damage induced by ulcerogenic manipulations. The severity of gastric mucosal lesions seen after administration of intracisternal thyrotropin-releasing hormone analogue, subcutaneous indomethacin, intragastric HCl, or intragastric ethanol was evaluated in OLETF and control LETO rats. Administration of thyrotropin-releasing hormone analogue, indomethacin, HCl, or ethanol significantly increased the severity of gastric mucosal lesions in OLETF rats compared with control LETO rats. These results suggest for the first time that gastric mucosal integrity may be impaired in OLETF rats. It is furthermore speculated that individuals that lack of CCK-A receptors are highly susceptible to gastric ulceration.

Cholecystokinin (CCK) is detected in pituitary tumors but its role remains unknown. On the hypothesis that CCK may facilitate the cell growth in pituitary tumors, we have examined the effect of CCK on cell growth using a rat pituitary tumor cell line, GH3, cultured in a serum-free, chemically defined medium. Addition of sulfated CCK-(26-33) (CCK-8) in two different concentrations (0.5 approximately 1 nM) caused a significant increase in the number of GH3 cells. The antagonist (1 microM) for CCK-B receptor, but not CCK-A receptor, significantly inhibited the number of GH3 cells. Northern blot analysis revealed a significant expression of CCK-B receptor mRNA in GH3 cells, but not in normal rat pituitary glands. In addition, immunoreactive CCK/gastrin was detected by RIA in the GH3 cell extracts as well as the serum-free culture medium. In GH3 cell extracts, both CCK-8 and gastrin like peptides were identified by gel chromatography. These findings provided the first evidence for an autocrine/paracrine role of CCK and gastrin on stimulation of GH3 cell growth through the CCK-B receptor.

Gastrin stimulates gastric acid secretion through direct activation of CCK-B/gastrin receptors on parietal cells and indirectly through release of histamine from ECL cells. Cholecystokinin (CCK) is structurally closely related to gastrin and shares high affinity for CCK-B/gastrin receptors. In contrast to gastrin, CCK also recognizes CCK-A receptors. While CCK appears to be a negative regulator of gastric acid secretion and postprandial release of gastrin in the normal human gastrointestinal tract, its impact on the pathogenesis of acid hypersecretion in Helicobacter pylori-infected individuals remains uncertain. This article will review the endocrine and paracrine regulatory pathways which are activated by CCK/gastrin peptides and which appear relevant in the pathogenesis of peptic ulcer disease in man.

The effects of the gastrointestinal hormones secretin and cholecystokinin (CCK) were investigated on rat portal-vein mechanical activity. Doses of 0.03-0.60 units/ml Boots secretin; 0.18-0.60 units/ml pure, natural secretin; and 0.12-0.60 units/ml CCK elicited dose-dependent decreases in amplitude and simultaneous increases in frequency of spontaneous contractions that were unaffected by alpha-or beta-adrenergic or cholinergic blocdade. These effects were mimicked by isoproterenol, theophylline, and cyclic AMP, and all agents tested were also effective in reducing K-or norepinephrine-induced tension. Additionally, subthreshold doses of CCK and secretin elicited responses when given in combination, and the effects of the hormomes at all doses were potentiated by theophyline, indicating an interaction berween these hormones on rat portal vein thatmay involve cyclic AMP. It is suggested that part of the mechanism of hormone-induced mesenteric vasodilatation may involve a direct relaxing effect of the hormones on vascularsmooth muscle.

Phospholipase C is involved in the insulinotropic effect of carbachol (CCh) and cholecystokinin octapeptide (CCK-8). The involvement of the type of G protein was investigated in rat pancreatic islets. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; a nonhydrolyzable GTP analogue) increased insulin release in electrically permeabilized islets. Both CCh and CCK-8 increased the GTP gamma S effect indicative of an involvement of G proteins. Pretreatment of the islets with pertussis toxin (PT) impaired the CCh-induced insulin secretion in the presence of 3.0 mM glucose and inhibited the stimulatory CCh effect on inositol 1,4,5-trisphosphate (IP3) levels at low and high glucose. In contrast to CCh, the CCK-8 effect on both insulin release and IP3 levels of islets was not modified by a PT pretreatment at various glucose concentrations. Two types of experiments indicate the type of G protein involved: first, long-term agonistic stimulation by either CCh or CCK-8 led to a downregulation of alpha o and alpha q/11, respectively; second, introduction of specific anti-alpha o or -alpha q/11 antibodies into electrically permeabilized islets nearly completely abolished the effects of CCh and CCK-8, respectively. The data indicate that both CCh and CCK-8 act as insulinotropic agents via the phospholipase C system; in the effect of CCh the PT-sensitive alpha o and in the effect of CCK-8 the PT-insensitive alpha q/11 is involved.

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCKA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCKCCKA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCKCCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

A series of nitrogen-containing benzophenone analogues were synthesized by Mannich reaction and evaluated for the inhibition of pro-inflammatory cytokines, TNF-alpha and IL-6. DPPH (1,1-diphenyl-2-picryl hydrazine) radical scavenging activity and its kinetics were studied to determine the antioxidant potential of the test samples. All the synthesized compounds exhibited promising activity against IL-6 in a range of 81-89%, 09-42% at 10 and 1 microM, respectively, concentration. Exceptionally, the compound 20e was observed to be an effective inhibitor of TNF-alpha (54%) and IL-6 (97%), (47%) at 10 and 1 microM concentrations with minimum toxicity (22%) against CCK-8 cells. With few exceptions, all other compounds were found to be excellent inhibitors of IL-6 and moderate to excellent inhibitors of TNF-alpha, however the toxicity profiles of these compounds need to be ameliorated in further optimization studies. Amongst the tested compounds, 16a, 17g, 18f, 18g, 19g and 20e were found to possess significant antioxidant activity.

The selective cholecystokinin (CCK)-B-receptor agonist and antagonist, BC 264 and L 365260, respectively, and the CCK-A-receptor antagonist, L 364718, were used to investigate the possible involvement of different classes of CCK receptors in the control of food intake induced by exogenous CCK octapeptide (CCK-8) in the cat with gastric fistula. Intravenous infusion of CCK-8 dose dependently inhibited milk intake under sham-feeding conditions, maximal inhibition reaching 52 +/- 7% (P less than 0.001) with 0.88 nmol.kg-1.h-1. L 364718 prevented this inhibition, whereas L 365260 was ineffective over the dose range tested. The reversal effect of L 364718 on 0.88 nmol.kg-1.h-1 CCK-8-induced inhibition of milk intake was observed at doses as low as 0.44 nmol.kg-1.h-1. The selective CCK-B-receptor agonist, BC 264, in doses ranging from 0.88 to 7 nmol.kg-1.h-1, had no effect on milk intake under sham-feeding conditions, although it dose dependently stimulated gastric acid output. Furthermore, neither L 364718 nor L 365260 (88 nmol.kg-1.h-1 iv) stimulated milk intake when given in the absence of CCK-8. We conclude that exogenous CCK-8 causes satiety in the cat through activation of peripheral CCK-A receptors.

We studied the importance of cholecystokinin (CCK) system in the regulation of thyrotropin (TSH) and prolactin (PRL) secretion in male rats. To this end, we tested the effects of both unselective CCK agonists CCK-8 and caerulein, and CCK-B selective agonists CCK-4 and pentagastrin as well as the selective CCK antagonists (devazepide and L-365,260) at wide dose-ranges on the cold-stimulated and TRH-induced TSH and PRL secretion. Caerulein, given s.c. 15 min before sacrifice, decreased TSH levels at 5 micrograms/kg. In time course-studies, the maximum inhibition was seen at 15 min but the effect lasted at least 30, but less than 60 min. Also CCK-8 decreased TSH levels at the doses of 20 and 50 micrograms/kg at 15 min. Devazepide and L-365,260 did not affect TSH or PRL levels at any dose. The effect of caerulein (5 micrograms/kg) was antagonized by devazepide, a CCK-A antagonist, at 100 micrograms/kg, but not by a CCK-B antagonist L-365,260 tested at a wide dose range. PRL levels were not affected by any treatment. Caerulein (5 micrograms/kg), given at the same time as TRH (500 ng/kg), inhibited the TRH-induced TSH levels at 15 min, but not at 30 or 60 min. CCK-8 (50 micrograms/kg), CCK-4 (100 micrograms/kg) and pentagastrin (500 micrograms/kg) did not affect the TRH-induced TSH secretion. The results probably indicate that CCK-A receptor stimulation inhibits TSH secretion at the level of the anterior pituitary gland. PRL levels in male rats are not affected by CCK system.

A drug design strategy to non-peptide small molecule antagonists of neuropeptides is described that targets the molecular diversity which exists in the 'privileged' data set of the physico-chemical properties represented by the side-chains of the 20 genetically encoded amino acids. The strategy is exemplified by the design of a selective and high affinity cholecystokinin CCK-A antagonist PD 140548, CCK-B antagonist CI-988 (formerly PD 134308) tachykinin NK-1 antagonist PD 154075 and NK-2 antagonist Cam-2291. The NK-3 antagonists, PD 157672 and the non-peptide PD 161182, were developed from an information-rich dipeptide library constructed from 256 N-protected dipeptides and 64 hydrophobic biased dipeptides.