The Ewing family of tumors is a group of highly malignant tumors that mainly arise in bone and most often affect children and young adults in the first two decades of life. Despite the use of multimodal therapy, the long-term disease-free survival rate of patients with Ewing tumors is still disappointingly low, making the discovery of innovative therapeutic strategies all the more necessary. We have recently shown that cholecystokinin (CCK), a neuroendocrine peptide, involved in many biological functions, including cell growth and proliferation, is a relevant target of the EWS/FLI1 oncoprotein characteristic of Ewing tumors. CCK silencing inhibits cell proliferation and tumor growth in vivo, suggesting that CCK acts as an autocrine growth factor for Ewing cells. Here, we analyzed the impact of two CCK receptor antagonists, devazepide (a CCKantagonist) and L365 260 (a CCKantagonist), on the growth of Ewing tumor cells. Devazepide (10 micromol/l) inhibited cell growth of four different Ewing tumor cells in vitro (range 85-88%), whereas the effect of the CCKantagonist on cell growth was negligible. In a mouse tumor xenograft model, devazepide reduced tumor growth by 40%. Flow cytometry experiments showed that devazepide, but not L365 260, induced apoptosis of Ewing tumor cells. In summary, devazepide induces cell death of Ewing tumor cells, suggesting that it could represent a new therapeutic approach in the management of Ewing's tumor patients.

Gastric and sphincter motility evoked by intravenous injection of CCK-8 were investigated in urethane-anesthetized rats. Digital ultrasonomicrometry was used to monitor pyloric (PYL), antral (ANT), corpus (COR), and lower esophageal sphincter (LES) movements while simultaneously measuring intragastric pressure (IGP) and, in some experiments, subdiaphragmatic intraesophageal pressure (sIEP). Intracrystal distances (ICD) were measured continuously between pairs of piezoelectric crystals affixed to the serosa of PYL, ANT, COR (circular and longitudinal), and LES. Consecutive intravenous injections of CCK-8 (0.3, 1, and 3 microg/kg) at 30-min intervals caused dose-dependent simultaneous tonic contractions of PYL and ANT, LES opening, and drops in IGP with peak changes at 3 microg/kg of -17.9 +/- 2.1, -7.7 +/- 2.5, 6.5 +/- 1.4, and -29.2 +/- 3.8%, respectively, whereas intravenous saline had no effect. Rhythmic contractile activity was inhibited by CCK-8. COR responses were not significantly different from vehicle controls for most metrics, and the direction of response for circular COR varied between preparations, although not for repeated trials in a single preparation. During the LES response to CCK-8, sIEP rose in parallel with drops in IGP, indicating formation of a common cavity. Recovery of LES ICD after intravenous CCK occurred more rapidly than recovery of PYL ICD, suggesting the importance of preventing simultaneous patency of gastroesophageal and gastroduodenal passages. The CCK(A) receptor antagonist devazepide (500 microg/kg intravenous) inhibited motion responses evoked by intravenous CCK-8. These data revealed CCK-8-induced gastric and sphincter activity consistent with retropulsion of gastric content.

1. Dipsacus saponin C (DSC) administered intracerebroventricularly (i.c.v.) showed an antinociceptive effect in a dose-dependent (from 3.75 to 30 micrograms) manner as measured by the tailflick assay. The antinociception induced by DSC at the dose of 30 micrograms was maintained at least 1 h. 2. Sulfated cholecystokinin (CCK, from 0.1 to 0.5 ng); muscimol (a GABAA receptor agonist, from 50 to 200 ng); MK-801 [(+/-)-5-methyl-10, 11-dihydro-5H-dibenzo (a,d) cyclohepten-5, 10-imine maleate, from 0.1 to 1 microgram], a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist; or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, from 0.1 to 0.5 microgram), a non-NMDA receptor antagonist, injected i.c.v. significantly reduced the inhibition of the tail-flick response induced by DSC (30 micrograms) administered i.c.v. However, naloxone (an opioid receptor antagonist, 2 micrograms) or baclofen (a GABAB receptor antagonist, 10 ng) did not affect the inhibition of the tail-flick response induced by DSC. 3. The intrathecal (i.t.) injection of yohimbine (an alpha 2-adrenergic receptor antagonist, from 5 to 20 micrograms) and methysergide (a serotonin receptor antagonist, from 5 to 20 micrograms) but not naloxone (from 2 to 8 micrograms), significantly attenuated inhibition of the tail-flick response induced by DSC (30 micrograms) administered i.c.v. 4. Our results suggest that DSC has an antinociceptive effect when it is administered supraspinally and GABAA, NMDA and non-NMDA receptors, but not opioid and GABAB receptors located at the supraspinal level, may be involved in DSC-induced antinociception. Furthermore, DSC administered supraspinally may produce antinociception by stimulating descending alpha 2-adrenergic and serotonin pathways but not the opioidergic pathway.

The design, synthesis, and structure-activity relationships (SAR) for the development of selective dipeptoid ligands for both of the cholecystokinin (CCK) receptor subtypes CCK-A and CCK-B are described. The SAR developed is used to design a ligand with equal nanomolar binding affinity for both the CCK-A and CCK-B receptors. Example compounds such as [1R-[1 alpha[R*(R*)],2 beta]]-4-[[2-[[3-(1H-indol-3-yl)- 2-methyl-2-[[[(2-methylcyclohexyl)oxy]carbonyl]amino]-1- oxopropyl]-amino]-1-phenylethyl]amino]-4-oxo-butanoic acid (24c), (1R-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy] carbonyl]-D-tryptophyl]-L-3-(phenylmethyl)-beta-alanine (28i), and N-[alpha-methyl-N-[(tricyclo[3.3.1.1]dec-2-yloxy) carbonyl]-D-tryptophanyl]-L-3-(phenylmethyl)-beta-alanine (30m) are CCK-B selective compounds having CCK-B binding affinities of IC50 = 3.9, 0.34, and 0.15 nM with a CCK-A/CCK-B ratio of 464, 53, and 170, respectively. Other compounds such as (1R-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy]carbonyl]- L-tryptophyl]-D-3-(phenylmethyl)-beta-alanine (281) and N-(alpha-methyl-N-[(tricyclo[3.3.1.1]dec-2-yloxy)carbonyl]-L - tryptophyl]-D-3-(phenylmethyl)-beta-alanine (30p) are CCK-A-selective compounds having CCK-A binding affinities of IC50 = 7.9 and 2.82 nM with a CCK-A/CCK-B ratio of 0.007 and 0.01, respectively. Further to these, (1S-trans)-N-[alpha-methyl-N-[[(2-methylcyclohexyl)oxy] carbonyl]-D-tryptophyl]-L-3-(phenylmethyl)-beta-alanine (28h) is a mixed CCK-A/CCK-B ligand with a CCK-A binding affinity of IC50 = 3.9 nM and a CCK-B binding affinity of IC50 = 4.2, producing a CCK-A/CCK-B ratio of unity. The CCK-B selective compounds are shown to be antagonists in electrophysiological tests on the rat ventromedial nucleus of the hypothalamus with an equilibrium constant (Ke) value of 2.8 nM for 30m and are also shown to be anxiolytic in the mouse ligh/dark box test with a minimum effective dose of 0.01 mg/kg, sc, for 30m. The CCK-A selective compounds are also shown to be competitive antagonists by the inhibition of CCK-8S-evoked amylase secretion from pancreatic acinar cells with a Ke value of 16 nM for 30p. In electrophysiological tests on the rat dorsal raphé (an area rich in CCK-A receptors) 30p had a Ke value of 12.8 nM. The mixed CCK-A/CCK-B compound 28h showed antagonistic properties in both CCK-A and CCK-B models; thus it inhibited CCK-8S-evoked amylase secretion from pancreatic acinar cells and is anxiolytic in the light/dark box paradigm.(ABSTRACT TRUNCATED AT 400 WORDS)

Since the time of Freud, psychiatry has embraced the proposition that physiological and/or psychological stress precipitates various psychiatric disorders. To this effect, we propose that a continuum of liability obtains between stress, anxiety states and anorexia nervosa--a continuum which is grounded on a cytokine profile common to each of these conditions. For example, the biological response to stress, anxiety states and anorexia nervosa includes the elevation of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), and downregulation of interferon-gamma (IFN-gamma). Sustained elevation of IL-1 beta and TNF-alpha dysregulates both somatostatin and insulin secretion, the latter of which influences regional cerebral blood flow (rCBF) and brain energy metabolism. In addition, IL-1 beta and TNF-alpha influence the expression of certain crucial neuropeptides, which are known to be associated with anxiety states and anorexia nervosa. These neuropeptides include: beta-endorphin, cholecystokinin (CCK), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). beta-endorphin effects glucose metabolism in the limbic system, CCK increases the release of beta-endorphin from the anterior pituitary, NPY is a powerful anxiolytic that regulates beta-endorphin and insulin, while VIP indirectly regulates the expression of TNF-alpha through the inhibition of interleukin-4 (IL-4).

OBJECTIVE

Metabolism of long-chain fatty acids within the duodenum leads to the activation of duodenal mucosal protein kinase C (PKC)-δ and the cholecystokinin (CCK)-A receptor to lower glucose production through a neuronal network. However, the interfunctional relationship between duodenal PKC-δ and CCK remains elusive. Although long-chain fatty acids activate PKC to stimulate the release of CCK in CCK-secreting cells, CCK has also been found to activate PKC-δ in pancreatic acinar cells. We here evaluate whether activation of duodenal mucosal PKC-δ lies upstream (and/or downstream) of CCK signaling to lower glucose production.

METHODS

We first determined with immunofluorescence whether PKC-δ and CCK were colocalized within the duodenal mucosa. We then performed gain- and loss-of-function experiments targeting duodenal PKC-δ and the CCK-A receptor and evaluated the impact on changes in glucose kinetics during pancreatic (basal insulin) clamps in rats in vivo.

RESULTS

Immunostaining of PKC-δ was found to colocalize with CCK in the duodenal mucosa. Intraduodenal coinfusion of either the CCK-A receptor antagonist MK-329 or CR-1409 with the PKC activator negated the ability of duodenal mucosal PKC-δ activation to lower glucose production during the pancreatic clamps in normal rats. Conversely, molecular and pharmacological inhibition of duodenal PKC-δ did not negate the ability of the duodenal CCK-A receptor agonist CCK-8 to lower glucose production, indicating that activation of duodenal PKC-δ lies upstream (and not downstream) of CCK signaling. Finally, intraduodenal PKC activator infusion failed to lower glucose production in rats with high-fat diet-induced duodenal CCK resistance.

CONCLUSIONS

In summary, activation of duodenal PKC-δ leads to the stimulation of CCK release and activation of the CCK-A receptor signaling axis to lower glucose production in normal rats, but fails to bypass duodenal CCK-resistance in high fat-fed rats.

The display of lordosis behavior has been correlated with the estrogen-induced expression of cholecystokinin (CCK) and enkephalin within the limbic-hypothalamic circuit. These neuropeptides have opposing effects on lordosis; for example, in the medial preoptic nucleus, CCK facilitates and opiates inhibit lordosis. Antisense oligodeoxynucleotide blockade of receptor expression indicated that CCK modulates lordosis in the medial preoptic nucleus through the CCK(A)-receptor. Sequence-specific antibodies directed against delta- and mu-opiate receptor proteins labeled fibers in the medial preoptic nucleus. Estrogen treatment of ovariectomized rats or etorphine (a nonselective opiate agonist) treatment altered the appearance of the immunoreactivity from a diffuse pattern to one of distinctly stained mu-opiate receptor immunoreactive cells and varicose fibers in the medial preoptic nucleus. Such a pattern of staining reflects an internalization of mu-opiate receptors following agonist stimulation. This type of internalization has been used as an indication of synaptic activity. The distribution of receptor internalization surrounds the distribution of CCK cells in the medial preoptic nucleus, suggesting that endogenous opioid peptides may modulate estrogen-induced CCK mRNA expression. Interestingly, nonselective and delta-opiate receptor selective antagonists potentiated the estrogen-induced CCK mRNA expression in the medial preoptic nucleus. Together, these results suggest that endogenous opioid peptides may modulate the estrogenic upregulation of CCK mRNA expression and demonstrate an important level of regulation of gene expression in which synaptic activity modifies hormonal input.

Recent studies in genetically obese and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats suggest defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions such as pancreatic juice, protein, and gastric acid secretion. The present studies were undertaken to further examine CCK-A receptor gene expression and CCK-A receptor-mediated biological functions in the pancreas, stomach, and brain of OLETF rats. Expression of the CCK-A receptor gene could not be detected in the stomach, pancreas and brain by the reverse-transcription polymerase chain reaction (RT-PCR) method and Southern blotting of the PCR products. Southern blot analysis of genomic DNA from OLETF and control Long-Evans Tokushima Otsuka (LETO) rats with CCK-A receptor fragment as a probe revealed different restriction bands. Expression of the CCK-B receptor gene was observed in the stomach, pancreas, and brain in both OLETF and LETO rats by the RT-PCR method, with expression of the CCK-B receptor gene markedly enhanced in OLETF rats compared with that in LETO rats. Consistent with the defect of CCK-A receptor gene expression, CCK-A receptor-mediated biological functions were not observed in these organs. Perfused exocrine and endocrine pancreas of OLETF rats were insensitive to CCK stimulation but not to carbamylcholine stimulation. Basal gastric acid and pepsinogen secretions in OLETF rats were higher than in LETO rats. OLETF rats showed a significantly higher average daily food intake, gained body weight faster, and were heavier than LETO rats. The present study confirmed that OLETF rats have CCK-A receptor gene anomalies and demonstrated deficient CCK-A receptor-mediated biological function in the pancreas, stomach, and brain.

The mouse cholecystokinin type-A receptor (CCK(A)R) gene was cloned and sequenced, and the exon/intron boundaries were determined by cDNA cloning. The gene, approximately 10 kb in length, contains the entire coding region, and consists of five exons. The deduced amino acid sequence was homologous with that of other species, with the exception of an additional DNA sequence encoding 7 amino acids in exon 5. A region of the 5' end of exon 2 appeared to be alternatively spliced, and generated an isoform shorter by 52 bases. The shorter isoform may encode an 48 amino acid open reading frame due to frameshift of translation. These two mRNA isoforms were expressed equally in the mouse gallbladders.

Naturally occurring dipeptides, cholecystokinine (CCK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10-30 min with 40 mM-nitrite (pH 3.5, 37 degrees C), and the resultant products examined for mutagenicity in Salmonella typhimurium TA100. Specific mutagenicities (net revertants per mumol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/mumol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/mumol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/mumol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/mumol, respectively). The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/mumol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

The results of our investigations into the localization of Na+,K+-pump activity in pancreatic and parotid acinar cells and the effects of hormones and neurotransmitters on pump turnover can be integrated with data on other aspects of stimulus-response coupling to construct models of the neurohumoral control of protein, fluid, and electrolyte secretion (Fig. 23). In both tissues, Ca2+ and cyclic AMP serve as intracellular messengers. In pancreatic acinar cells, the Ca2+-dependent pathway activated by the occupation of CCK or cholinergic receptors provides the primary stimulus for digestive enzyme secretion. Cyclic AMP plays a comparatively minor role; VIP and secretin are much less effective stimulators of protein secretion. Conversely, cyclic AMP levels in parotid acinar cells, which are modulated primarily through occupation of beta-adrenergic receptors, are a major determinant of enzyme secretion. Activation of the Ca2+-dependent pathway by cholinergic or alpha-adrenergic agonists or substance P is less important. The presence of dual control processes in each gland suggests that the observed differences in effectiveness of cyclic AMP- versus Ca2+-dependent secretagogues may reflect not different mechanisms, but rather a shift in the relative emphasis placed on each pathway. This emphasis could conceivably result from subtle variations in the interaction between cellular protein kinases and phosphatases and their phosphoprotein substrates. Electrolyte secretion, on the other hand, appears to involve both discrete and common entities. In pancreatic acinar cells from rodent species, cholinergic or CCK receptor occupancy elicits a Ca2+-dependent increase in the open-state probability of nonselective cation channels in the basolateral plasma membrane. The resultant influx of Na+ and efflux of K+ is most probably the factor which activates Na+, K+-pumps. Based on electron probe studies of the effects of cholinergic agonists on acinar cell Na+ and K+ contents discussed earlier, a transient reduction in the intracellular K+/Na+ ratio of up to 4-fold may occur. A shift of this magnitude in the cytoplasmic microenvironment of the Na+, K+-pump clearly would have a stimulatory influence (see discussion by Jorgensen, 1980). In addition, Ca2+ itself may have direct effects on Na+,K+-pump activity. Calcium at levels much above 1 microM progressively inhibits Na+,K+-ATPase activity (Tobin et al., 1973; Yingst and Polasek, 1985). In unstimulated guinea pig pancreatic acinar cells, Ca2+i measured by quin-2 fluorescence was 161 +/- 13 nM (Hootman et al., 1985a) which increased to a maximal concentration of 803 +/- 122 nM following CCh stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of cholecystokinin octapeptide (CCK-8) were investigated in CACCK-8 (100 nM) produced slight depolarizaton (2.1 +/- 0.3 mV) and reduced the amplitude of afterhyperpolarization following a train of spikes. CCK-8 (10 nM-1 microM) concentration-dependently reduced the amplitude of afterhyperpolarization. CCK-4, a selective agonist for CCK(B) receptors, also attenuated the amplitude of afterhyperpolarization. CCK-8-induced suppression was completely abolished by (+)L-365,260, a selective CCK(B) receptor antagonist, but not by (-)L-364,718, a selective CCK(A) receptor antagonist. Similarly, CCK-8 reduced the tail currents following a depolarizing pulse. The tail currents were characterized as Ca2+-activated K+ currents. When neurons were held at a holding potential of -40 mV, CCK-8 elicited inward currents with a reduction of membrane conductance. This current had a relatively linear current voltage relationship and was reversed in polarity at membrane potentials close to the K+ equilibrium potential, suggesting that CCK-8 decreases leak K+ currents. Moreover, voltage-activated Ca2+ currents were partially blocked by CCK-8, and this effect was enhanced by intracellular application of GTPgammaS (300 microM) or a protein phosphatase inhibitor, okadaic acid (100 nM), and attenuated by GDPbetaS (300 microM) or a protein kinase inhibitor, staurosporin (400 nM). In acutely-prepared hippocampal slices from neonatal rats, CCK-8 also depolarized CACCK-8 increases neuronal excitability by suppressing leak K+ currents and Ca2+-activated K+ currents in CACCK(B) receptors.

Cholecystokinin (CCK)/gastrin receptors were characterized in calf pancreatic plasma membranes from newborns, 28- and 119-day-old milk-fed preruminants, and 119-day-old weaned ruminants. Scatchard analysis of [125I]Bolton-Hunter reagent-[Thr28,Nle31]CCK-(25-33) binding indicated two classes of binding sites: high affinity sites exhibited significant higher affinity and binding capacity (P < 0.05) in 119-day-old ruminants than in 119-day-old preruminants (Kd = 0.13 +/- 0.02 vs. 0.35 +/- 0.08 nM; binding capacity (Bmax) = 53 +/- 12 vs. 18 +/- 5 fmol/mg protein). Pharmacological analysis using selective agonists and antagonists indicated the expression of the CCK-A receptor at birth, whereas the CCK-B receptor predominated at postnatal stages. At all stages, the binding was inhibited by guanosine 5'-[gamma-thio]triphosphate. Binding site identification by photoaffinity labeling showed that at birth, the labeling occurred mainly on a 78- to 96-kilodalton (kDa) component. In milk-fed animals, aged 28 and 119 days, two membrane-binding components were labeled at 78-96 and 43-52 kDa. In 119-day-old ruminants, labeling occurred mainly on a 40- to 47-kDa protein. Deglycosylation by endo-beta-N-acetylglucosaminidase-F of the 40- to 47- and 43- to 52-kDa components resulted in the formation of a 37-kDa membrane protein. Consequently, this study demonstrated 1) the differential expression of CCK-A and -B receptors in developing calf pancreas, 2) the predominance of CCK-B receptors in normal pancreas, and 3) the maturation of CCK-B receptors during the weaning period, which includes the glycosylation level. These results suggest that CCK may play a predominant role during the early postnatal development, while gastrin and CCK-B receptors can function progressively to regulate proliferation and exocrine secretion in the calf pancreas, especially from the weaning period.

Gastrin (cholecystokinin type B (CCK-B)) receptor antagonists may help to elucidate the physiological role of gastrin, have therapeutic potential as acid antisecretory drugs, and may be of use as adjuvant therapy for gastrin sensitive tumours. In binding studies, the gastrin receptor antagonist PD-136,450 had at least 1000 fold greater affinity for gastrin (CCK-B) than CCK-A receptors. In this study the biological activity of PD-136,450 was evaluated in conscious and anaesthetised rats. PD-136,450 antagonised gastrin stimulated acid secretion after subcutaneous (IC50: 0.28 mumol/kg; conscious rats) and intravenous (IC50: 0.17 mumol/kg; anaesthetised rats) administration. In basal secreting fistula animals, the compound stimulated acid output to 30 (5)% of the maximal response to gastrin. Stimulant activity was not caused by gastrin release. As an agonist PD-136,450 was about 350 times less potent than gastrin-17 on a molar basis. In addition, PD-136,450 was a powerful agonist of pancreatic secretion in anaesthetised rats. The specific gastrin antagonist L-365,260 inhibited the (partial) agonist activity of PD-136,450 in the stomach and the specific CCK-A receptor antagonist L-364,718 inhibited the agonist activity of PD-136,450 in the pancreas. It is concluded that the agonist effect of PD-136,450 is mediated via interaction with the gastrin (CCK-B) receptor in the stomach and the CCK-A receptor in the pancreas.

Two receptors for cholecystokinin (CCK) have been isolated which also bind gastrin: CCK-A type and CCK-B type, both are coupled to phospholipase C (PLC) activation. However, identification of the "true" gastrin receptor remains controversial. We determined which CCK receptor mediated the trophic effect of gastrin on human colon cancer cells (LoVo). LoVo cells lack mRNA for either CCK receptor by Northern hybridization. Gastrin stimulated cyclic AMP production, not PLC activity, in LoVo cells. The trophic effect was not blocked by receptor antagonists for CCK-A (L364,718) or CCK-B (L365,260). The gastrin receptor pharmacology on LoVo cells and the lack of appropriate transcripts suggest that gastrin stimulated growth of these cells by a receptor other than CCK-A or CCK-B type and there likely exists another receptor for gastrin.

Although it is known that panic attacks are triggered by the cholecystokinin fragment CCKCCK receptors in various adaptive processes such as emotion, memory and anxiety has yet to be demonstrated. With this aim, we have investigated the biochemical and pharmacological effects resulting from the administration of BC264, a highly potent and selective CCK-B agonist able to cross the blood-brain barrier. Very low doses of BC264 (microg/kg i.p.), increased the exploration of animals submitted to an unknown territory but were devoid of anxiogenic properties in the elevated plus maze. BC264 increased locomotion and rearings of rats newly placed in an open field and improved their spontaneous alternation in a Y-maze. The use of vagotomized animals showed that the increased alternation induced by BC264 did not require an intact vagus nerve, unlike the locomotor activation. These behavioural effects, prevented by the prior i.p. administration of the CCK-B antagonist L-365,260 but not by the CCK-A antagonist L-364,718, were shown to depend on dopaminergic systems, since they were blocked by D1 (SCH23390, 25 microg/kg i.p.) or D2 (sulpiride, 50 or 100 mg/kg i.p.) antagonists. In addition, bilateral perfusion in freely moving rats of BC264 at pharmacologically active doses, using a newly designed microdialysis system, was found to increase the extracellular levels of DA, DOPAC and HVA in the anterior part of the nucleus accumbens. These results show that activation of CCK-B receptors by BC264 does not produce anxiogenic-like effects but appears to improve motivation and attention, whereas other CCK-B agonists such as BocCCKCCK-B receptor, could account for these differential effects.

In the present study we have examined the interaction between the selective cholecystokinin (CCK)A and CCKB receptor antagonists, devazepide and L365-260 on morphine conditioned place preference (CPP). Using an unbiased procedure, morphine (1.5 mg/kg) produced a reliable CPP which was observed irrespective of the conditioning compartment type. Pretreatment with devazepide (0.001-0.01 mg/kg s.c.) produced a dose related attenuation of this response. At higher doses (0.1-1 mg/kg) this antagonism became variable and dependent on the training compartment with blockade only observed when conditioning was to the white/rough textured environment. This profile has also been reported for the serotonin (5-HT)3 receptor antagonist ondansetron. The CCKB antagonist L365-260 (0.000001-0.01 mg/kg) failed to antagonize the morphine CPP, if anything a mild potentiation was observed. To study this further we examined the interaction between L365-260 (0.01 mg/kg) and a subthreshold dose of morphine (0.3 mg/kg). At these doses neither drug elicited CPP, however when co-administered a significant CPP was recorded. Finally, L365-260 at 1 mg/kg induced a mild but significant CPP when administered alone. These results suggest a differential role of CCK receptor subtypes on reward-related behaviour and complement previous studies suggesting bimodal effects of CCK systems on mesolimbic dopamine function.

Cholecystokinin (CCK), a neurotransmitter in the central nervous system (CNS), co-exists in a large portion of A10 dopamine neurons to exert some effect on dopamine behavior. The aim of this study was to determine whether any association exists between the genotype of CCK gene promoter regions (-45C/T and -196G/A) and suicidal behavior. Genotypes and allele frequencies of CCK -45C/T and -196G/A were analyzed using polymerase chain reaction (PCR) followed by single-strand conformational polymorphism (SSCP) analysis on the genomic DNA from selected suicide victims (N=154) and from control subjects (N=328). Statistical analysis was performed using the Mantel-Haesnzel chi2-test and multiple logistic regression analysis with distinction of gender. An association between CCK -196G/A polymorphism and suicidal behavior in Japanese males was confirmed by statistical analysis (Odds ratio: 3.462, 95% CI: 1.128-10.626, P=0.038 by multiple logistic regression analysis). However, a significant association between CCK -196G/A polymorphism and suicidal behavior was not discovered in females. The polymorphism of the CCK gene promoter region was found to represent a susceptibility factor for suicidal behavior in Japanese males.

1. The relationships between cytosolic Ca2+ ([Ca2+]cyt; expressed as a fluorescence ratio at 400 nm and 500 nm using Indo-1) and contractile force was examined in strips of circular smooth muscles of canine gastric antrum. Rhythmic increases in [Ca2+]cyt were observed and contractions were biphasic. 2. In most muscles (70%), the amplitude of the second phase of the Ca2+ transient was less than or equal to the first phase of the Ca2+ transient, but the second phase of the contraction was much smaller than the first phase, suggesting a decrease in Ca2+ sensitivity during the second contractile phase. In 30% of muscles, the amplitude of the second phase of the Ca2+ transient was 2- to 3-fold greater than the first phase. In these muscles, the second phase of contraction was 10-fold greater than the first phase of contraction. Thus, a non-linear relationship between [Ca2+]cyt and force greatly amplifies force development when [Ca2+]cyt exceeds a threshold level. 3. Acetylcholine (ACh, 0.3-1 microM) increased the amplitudes of Ca2+ transients and basal [Ca2+]cyt between phasic contractions. The increase in basal [Ca2+]cyt did not cause tone to develop. ACh increased the amplitude of Ca2+ transients 2- to 3-fold and this was associated with a 15 to 20-fold increase in the force of phasic contractions. Pentagastrin (0.5 nM) and cholecystokinin octapeptide (CCK, 40 nM) had similar effects on Ca2+ transients and phasic contractions. 4. Bay K 8644 (0.1 microM) and TEA (5 mM) also increased the amplitudes of Ca2+ transients by 2- to 3-fold and phasic contractions by 15- to 30-fold. There was no significant difference observed between the [Ca2+]cyt-force relationships in the presence of agonists (i.e. ACh, pentagastrin and CCK) or when [Ca2+]cyt was increased by Bay K 8644 or TEA. These data suggest that agonist-dependent increases in Ca2+ sensitivity may not significantly regulate the [Ca2+]cyt-force relationship in antral muscles. 5. D600 (5 microM), added during stimulation with ACh (0.3 M), decreased [Ca2+]cyt and force without affecting the [Ca2+]cyt-force relationship. 6. Mechanisms exist for agonist-mediated enhancement of the Ca(2+)-force relationship. In alpha-toxin-permeabilized antrum, ACh (10 microM) with GTP (100 microM) or GTP gamma S (100 microM) increased the Ca(2+)-induced contraction at clamped levels of Ca2+. Phorbol 12,13-dibutyrate (PDBu, 10 microM) also increased the contractile force at a given level of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Vagal CCK-A receptors (CCKARs) and leptin receptors (LRbs) interact synergistically to mediate short-term satiety. Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed by vagal afferent neurons. We sought to demonstrate that this neurotransmitter regulates CCK and leptin actions on short-term satiety. We also examined the signal transduction pathways responsible for mediating the CART release from the nodose ganglia (NG). ELISA studies coupled with gene silencing of NG neurons by RNA interference elucidated intracellular signaling pathways responsible for CCK/leptin-stimulated CART release. Feeding studies followed by gene silencing of CART in NG established the role of CART in mediating short-term satiety. Immunohistochemistry was performed on rat NG neurons to confirm colocalization of CCKARs and LRbs; 63% of these neurons contained CART. Coadministration of CCK-8 and leptin caused a 2.2-fold increase in CART release that was inhibited by CCK-OPE, a low-affinity CCKAR antagonist. Transfection of cultured NG neurons with steroid receptor coactivator (SRC) or phosphatidylinositol 3-kinase (PI3K) small-interfering RNA (siRNA) or STAT3 lentiviral short hairpin RNA inhibited CCK/leptin-stimulated CART release. Silencing the expression of the EGR-1 gene inhibited the CCK/leptin-stimulated CART release but had no effect on CCK/leptin-stimulated neuronal firing. Electroporation of NG with CART siRNA inhibited CCK/leptin stimulated c-Fos expression in rat hypothalamus. Feeding studies following electroporation of the NG with CART or STAT3 siRNA abolished the effects of CCK/leptin on short-term satiety. We conclude that the synergistic interaction of low-affinity vagal CCKARs and LRbs mediates CART release from the NG, and CART is the principal neurotransmitter mediating short-term satiety. CART release from the NG involves interaction between CCK/SRC/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways.

BACKGROUND

Leptin, a product of the ob gene controlling food intake, has recently been detected in the stomach and shown to be released by cholecystokinin (CCK) and to induce gastroprotection against various noxious agents, but it is not known whether centrally applied leptin influences gastric secretion and mucosal integrity.

OBJECTIVE

In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) on gastric secretion and gastric mucosal lesions induced by topical application of 75% ethanol.

METHODS

Several major series of Wistar rats were used in this study. The effects of leptin or CCK applied i.c.v. on gastric secretion were examined using conscious rats with gastric fistulas. For the studies on gastroprotection the following series of rats were used to determine the effects of: (A) leptin and CCK applied centrally on this protection and the blockade of CCK(A) with loxiglumide (30 mg/kg i.p.) and CCK(B) receptors with RPR 102681 (30 mg/kg i.p.); (B) cutting of vagal nerves; (C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.); (D) inhibition of calcitonin gene-related peptide (CGRP) receptors with CGRP(8-37) (100 microg/kg i.p.), and (E) suppression of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v. ) on ethanol-induced gastric lesions in rats with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR.

RESULTS

Leptin and CCK-8 (0.01-5 microg/kg i.c.v.) dose dependently attenuated gastric lesions induced by 75% ethanol; the doses reducing these lesions by 50% (ED(50)) were 0.8 and 1.2 microg/kg, respectively. The protective effects of leptin and CCK-8 applied i.c. v. were accompanied by a significant rise in plasma leptin level and an increase in GBF. Blockade of CCK(A) receptors with loxiglumide abolished the protective and hyperemic effects of CCK but not those of leptin, while RPR 10268, a specific antagonist of CCK(B) receptors, counteracted leptin-induced protection and the rise in the GBF but failed to influence those afforded by CCK-8. For comparison, pretreatment with peripheral CCK-8 or leptin (10 microg/kg i.p.) causing a similar rise in the plasma leptin level also significantly reduced gastric lesions induced by 75% ethanol. The protective and hyperemic effects of centrally administered leptin were abolished by vagotomy, producing a fall in plasma leptin levels, and significantly attenuated by sensory denervation with capsaicin, by pretreatment with the CGRP antagonist, CGRP(8-37), or with L-NAME. A strong signal for iNOS mRNA was recorded in the gastric mucosa of leptin- and CCK-8-treated animals, whereas cNOS mRNA was unaffected.

CONCLUSIONS

(1) Central leptin exerts a potent gastroprotective action at a dose that has no influence on gastric secretion; (2) this protection depends upon CCK(B) receptors, vagal activity and sensory nerves, and involves hyperemia probably mediated by NO, and (3) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide on the rat stomach.

The pancreatic gland has an enormous potential for growth and regeneration, mainly in rodents. These processes remain mostly under the control of the GI hormone cholecystokinin (CCK). The human pancreas however does not show proliferative properties after partial pancreatectomy, but research in this field has been scarce. Recent studies indicate that CCK might not be the expected trophic agent since its two receptors CCK(A) and CCK(B) were not found on human exocrine pancreas. Therefore, if human pancreas grows and regenerates, it has to be under the influence of some unknown trophic factors. Neuropeptides receiving much attention lately as regulators of pancreatic functions could be among the searched trophic agents. This presentation focus on neuropeptides growth potential: GRP-Bombesin, GABA, PP, PYY, Neurotensin, SP, VIP, PACAP, CGRP and galanin. Some neuropeptides have moderate effects on pancreatic enzymes and electrolytes secretion: SP, VIP, PACAP. However, their trophic effects remain unexplored except for GRP-bombesin and PACAP. PACAP preferentially exhibits its mitogenic and proliferative effects on the pancreatic acinar cells AR4-2J via tyrosine kinase, phospholipase D and ornithine decarboxylase activation but not through adenylate cyclase. The growth promoting action of GRP-bombesin is well documented on rodent's pancreas. However, recent studies indicate that this neuropeptide is potentially trophic for larger mammals' pancreas. Indeed, investigators recently documented that bombesin induced pancreatic regeneration in the pig after partial pancreatectomy through mitogen-activated protein kinases activation as do CCK-8 and caerulein on rat pancreas. Have we found the magic pancreatic trophic factor in large mammals? Further investigations will tell.

We have previously described a cholecysto-sphincter of Oddi reflex whereby sphincter of Oddi (SO) motility is mediated in part by the degree of gallbladder distension. Therefore, we tested the hypothesis that cholecystectomy alters the response of the SO to endogenous and exogenous hormonal stimulation. Eight months after sham laparotomy (n = 8) or cholecystectomy (n = 10), prairie dogs were anesthetized with alpha-chloralose. The common bile duct was cannulated distally with a side-hole, pressure-monitored catheter perfused with degassed water at 0.15 ml/min. The duodenum was cannulated distal to the SO to allow perfusion of the proximal 30 cm of intestine with 20 mmol/L sodium oleate at 0.4 ml/min. In animals undergoing sham laparotomy the gallbladder was cannulated, aspirated, and kept empty throughout the experiment. SO phasic wave frequency (F), amplitude (A), and baseline pressure were measured for 60 minutes before and during intraduodenal (ID) perfusion of sodium oleate and then for 60 minutes before and 30 minutes during intravenous (IV) infusion of cholecystokinin-octapeptide (CCK-OP) at 10 ng/kg/min. A SO motility index (MI) (MI = F X A) was calculated for each 10-minute period. Common duct diameter and resting SO motility were unaltered 8 months after cholecystectomy. In animals that had sham laparotomy ID infusion of sodium oleate reduced SO MI by 46% (p = 0.06) and 75% (p less than 0.05) at 30 and 60 minutes, respectively, whereas in animals that had cholecystectomy the reduction in SO MI was only 6% and 25% (p less than 0.05) during the same periods. In animals that had sham laparotomy IV CCK-OP increased the SO MI by 175% (p less than 0.05), but in the animals that had cholecystectomy IV CCK-OP increased SO MI by only 60% (no significance). These findings indicate that after cholecystectomy resting SO motility is unaltered, but the response to ID sodium oleate and to IV cholecystokinin is blunted. We suggest that cholecystectomy alters neural pathways that mediate the normal response of the SO to endogenous and exogenous hormonal stimulation.

Sprague-Dawley rats injected with a "physiological" dose of cholecystokinin octapeptide (CCK-8; 6 micrograms/kg ip) expressed c-fos immunoreactivity in the nucleus of the tractus solitarius (NTS) and the area postrema (AP) of the brain stem. Injection of the CCK-A antagonist L-364,718 30 min before CCK-8 injection eliminated c-fos expression in these regions. These findings support the hypothesis that CCK-8 induced c-fos expression is mediated by CCK-A receptors. We then tested whether a meal (Isocal) could activate c-fos, and, if so, whether this response could be eliminated by L-364,718. Ingestion of Isocal induced c-fos immunoreactivity in the NTS and AP. Meal-induced c-fos expression was not blocked by the CCK-A antagonist L-364,718. These findings demonstrate for the first time that a purely physiological nonnoxious stimulus, a meal, induces c-fos in the rat brain stem and indicate that feeding induces c-fos expression by a pathway that is largely, if not entirely, independent of CCK release.