Central cholecystokinin (CCK)-ergic neurotransmission has been implicated in the genesis of negative emotions. Most animal studies on the neurochemical background of CCK-induced anxiety have, up to date, exploited exploratory activity paradigms. The interaction of CCK with GABAergic inhibitory neurotransmission, mediated probably through CCK-B receptors, could be the neurochemical substrate for anxious type of exploratory behaviour. However, the CCK-A and CCK-B receptor-mediated interactions of this neuropeptide with mesencephalic dopaminergic regulation of motivation for locomotor activity have the potential to interfere with the behavioural outcome from routine exploratory activity tests. Systemic treatment with CCK receptor antagonists is likely to influence both GABA- and dopamine-linked CCK-ergic neurotransmission, and therefore their effects in exploratory activity tests should be interpreted with caution.

Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and "anti-opioid" by actions on pain-modulatory cells within the rostral ventromedial medulla (RVM). One consequence of activation of RVM CCK₂ receptors may be enhanced spinal nociceptive transmission; but how this might occur, especially in states of pathological pain, is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold after ligation of L₅/L₆ spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time-related increase in (prostaglandin-E₂) PGE₂ measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE₂ was approximately 5-fold and was observed at approximately 80 minutes post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-hydroxyindoleacetic acid (5-HIAA), a 5-hydoxytryptophan (5-HT) metabolite, as compared with controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT₃ antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE₂ and 5-HT in the spinal cord.

In this study, we used an in vivo anesthetized rat model to investigate the mechanisms responsible for duodenal acid-induced inhibition of gastric motility. Intraduodenal infusion of HCl produced a rate-dependent decrease in intragastric pressure. Infusion of HCl at 2 ml/h produced a physiological plasma secretin level and elicited a decrease in intragastric pressure of 3.0 +/- 0. 2 cmH20. Infusion of rabbit secretin antiserum reduced the acid-induced inhibition of gastric motility by 85 +/- 5%, suggesting mediation mainly by endogenous secretin. Administration of the cholecystokinin (CCK)-A antagonist MK-329 caused only a modest 10 +/- 3% reduction in gastric relaxation, whereas the serotonin antagonist ICS-205930 had no effect. In contrast, immunoneutralization with the secretin antibody caused only a 15% reduction in the relaxation evoked by a higher rate of HCl infusion (3 ml/h), whereas MK-329 and ICS-205930 caused a 20 +/- 4% reduction and no reduction, respectively. Bilateral truncal vagotomy or perivagal application of capsaicin completely abolished gastric relaxation in response to low rates (1-2 ml/h) of 0.1 N HCl infusion but only partially affected gastric relaxation in response to a higher infusion rate (3 ml/h). These observations indicate that multiple pathways mediate the duodenal acid-induced inhibition of gastric motility. At low rates of HCl infusion, gastric relaxation is mediated primarily by endogenous secretin, which acts through vagal afferent pathways. At higher rates of HCl infusion, gastric relaxation is mediated by endogenous secretin, CCK, and possibly by the direct action of HCl on vagal afferent pathways or yet unidentified neuropathways.

Resveratrol is a phytoalexin with strong antioxidant and anti-inflammatory effects reaching high concentrations in red wine. The aim of our study was to test the effects of resveratrol pretreatment on cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis in rats. Animals were divided into a control group, a group treated with CCK-8 and a group receiving 10 mg/kg resveratrol prior to CCK-8 administration. Resveratrol ameliorated the CCK-8-induced changes in the laboratory parameters, and reduced the histological damage in the pancreas. The drug failed to improve the pancreatic antioxidant state, but increased the amount of hepatic reduced glutathione and prevented the reduction of hepatic catalase activity. Resveratrol-induced inhibition of nuclear factor kappa B (NF-kappaB) activation or reduction of the pancreatic tumor necrosis factor-alpha (TNF-alpha) concentration could not be demonstrated. In conclusion, the beneficial effects of resveratrol on acute pancreatitis seem to be mediated by the antioxidant effect of resveratrol or by an NF-kappaB-independent anti-inflammatory mechanism.

1. The expression of c-fos mRNA in the rat hypothalamus was examined by in situ hybridization following systemic administration of cholecystokinin (CCK), a procedure known to activate magnocellular oxytocin neurons but not magnocellular vasopressin neurones. 2. Conscious male rats were given a single I.P. injection of 50 micrograms/kg CCK, c-fos mRNA signal was apparent in the supraoptic and paraventricular nuclei in rats killed 10 min after injection but not in uninjected or saline-(vehicle) injected rats. The density of c-fos mRNA at both sites was further elevated in rats killed 30 min or 60 min following injection, and was absent in rats killed 4 h after injection. 3. In the paraventricular nucleus the most dense expression of c-fos mRNA following CCK administration was in the medial, mainly parvocellular portion of the nucleus, in an area corresponding to the distribution of corticotrophin-releasing factor mRNA determined by in situ hybridization in adjacent sections. 4. The I.P. injection of CCK increased plasma oxytocin concentrations, measured by specific radioimmunoassay from 13 +/- 5 pg/ml in control rats to 107 +/- 9 pg/ml in the rats killed 10 min after injection, a similar response to that observed previously in urethane-anaesthetized rats. 5. In each of six urethane-anaesthetized rats, recordings were made from single neurones in the supraoptic nucleus, identified antidronomically as projecting to the posterior pituitary and identified electrophysiologically as putative oxytocin neurones. Following I.P. injection of 50 micrograms/kg CCK, the neurones increased their firing rate by a mean of 1.3 +/- 0.2 spikes/s averaged over the 10 min following injection. 6. From the appearance of c-fos mRNA in supraoptic neurones following CCK administration we conclude that this message is expressed in magnocellular oxytocin neurones, since vasopressin neuronal activity and vasopressin release is known to be unaffected by this stimulus, and since the supraoptic nucleus contains essentially only oxytocin neurones and vasopressin neurones. 7. We conclude that c-fos mRNA expression can be induced in supraoptic oxytocin neurones following brief and modest episodes of electrical activation, suggesting that c-fos may be involved in the gene regulation of these neurones under physiological conditions.

CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.

The current study has determined the ability of (pGlu-Gln)-CCK-8 to counter the development of diet-induced obesity-diabetes and examined persistence of beneficial metabolic effects in high fat and ob/ob mice, respectively. Twice daily injection of (pGlu-Gln)-CCK-8 in normal mice transferred to a high fat diet reduced energy intake (p < 0.001), body weight (p < 0.01), circulating insulin and LDL-cholesterol (p < 0.001) and improved insulin sensitivity (p < 0.001) as well as oral and intraperitoneal (p < 0.001) glucose tolerance. Energy intake, body weight, circulating insulin and glucose tolerance of (pGlu-Gln)-CCK-8 mice were similar to lean controls. In addition, (pGlu-Gln)-CCK-8 prevented the effect of high fat feeding on triacylglycerol accumulation in liver and muscle. Interestingly, (pGlu-Gln)-CCK-8 significantly (p < 0.001) elevated pancreatic glucagon content. Histological examination of the pancreata of (pGlu-Gln)-CCK-8 mice revealed no changes in islet number or size, but there was increased turnover of beta-cells with significantly (p < 0.001) increased numbers of peripherally located alpha-cells, co-expressing both glucagon and GLP-1. Beneficial metabolic effects were observed similarly in ob/ob mice treated twice daily with (pGlu-Gln)-CCK-8 for 18 days, including significantly reduced energy intake (p < 0.05), body weight (p < 0.05 to p < 0.01), circulating glucose (p < 0.05 to p < 0.01) and insulin (p < 0.05 to p < 0.001) and improved glucose tolerance (p < 0.05) and insulin sensitivity (p < 0.001). Notably, these beneficial effects were still evident 18 days following cessation of treatment. These studies emphasize the potential of (pGlu-Gln)-CCK-8 for the treatment of obesity-diabetes.

To assess the importance of triglyceride digestion products in producing satiety, we determined the effects of duodenal infusions of triolein, oleic acid, and oleic acid plus monoolein on meal patterns in ad libitum-feeding rats. Oleic acid and oleic acid plus monoolein inhibited feeding similarly; triolein's effect was delayed and fourfold less potent. We then used the type A cholecystokinin (CCK-A)-receptor antagonist devazepide to assess the importance of CCK in mediating the anorexia produced by oleic acid. Oleic acid (at 320, 440, and 640 mumol/h) inhibited 3-h intake dose dependently by 32, 56, and 75%, respectively. Devazepide (1 or 2 mg/kg) blocked the responses to the 320 mumol/h dose, but had little if any effect on responses to the larger doses. Devazepide (1 mg/kg) did block anorexic responses to 3-h cholecystokinin octapeptide infusions (3 and 10 nmol.kg-1.h-1 iv) that inhibited 3-h intake by 25 and 65%, respectively. Our results suggest that the satiety response to triolein is produced by the products of triolein digestion and that CCK plays a significant, indispensable role in mediating the satiety response to duodenal delivery of small but not large loads of oleic acid.

Peptones are potent stimulants of cholecystokinin (CCK) release in rats, both in vivo and ex vivo in a model of isolated vascularly perfused duodeno-jejunum preparation and in vitro in the intestinal CCK-producing cell line STC-1. The underlying mechanisms were here investigated with this cell line. Protein hydrolysates from various origins (meat, casein, soybean, and ovalbumin; 0.5-1%, wt/vol) dose dependently increased CCK release. Cephalosporin antibiotics, which mimic tripeptides, also stimulated the release of CCK over the concentration range 1-20 mM. The study of concentration dependence of cephalosporin uptake indicated a passive diffusion process at either pH 7.4 or pH 6.0, thus arguing against the involvement of a peptide transporter in CCK secretion. After pertussis toxin treatment (200 ng/ml; 5 h), the peptone- and cephalexin-induced CCK secretion was significantly reduced, suggesting the involvement of pertussis toxin-sensitive heterotrimeric G protein(s) in the secretory activity of STC-1 cells. Consistent with this was the identification by Western blot of G(i2)alpha, G(i3)alpha, and G(o)alpha immunoreactivities in STC-1 cell extracts. Additionally, peptones and cephalexin increased the cellular content in inositol phosphates, whereas a mild increase in cAMP content was restricted to peptone-treated cells. Protein kinase A or C inhibition did not modify peptone- or antibiotic drug-evoked CCK release. The extracellular Ca2+ chelator EGTA (500 microM) and the intracellular Ca2+ chelator BAPTA-AM [1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; 20 microM] abolished the peptone- and antibiotic drug-induced CCK release. Nifedipine and verapamil (10 microM) reduced by about 50% the CCK secretion evoked by these two secretagogues. In conclusion, peptones and some cephalosporins are potent stimulants of CCK release in the STC-1 cell line. The cellular mechanisms involve pertussis toxin-sensitive G protein(s) and are dependent on Ca2+ availability. We suggest that the STC-1 cell line is a useful model to study the molecular basis of peptone-induced CCK secretion.

The high sensitivity of pentagastrin stimulation in detecting primary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in >90% of MTCs but in a high percentage of small cell lung cancers and potentially a variety of gastrointestinal adenocarcinomas. In a pilot study, we have demonstrated the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tissues in vivo in animals and patients (T. M. Behr et al., Eur. J. Nucl. Med., 25: 424-430, 1998). The aim of the present study was to systematically optimize, in a preclinical model, suitable radioligands for targeting CCK-B receptors in vivo. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the COOH-terminal CCK-receptor binding tetrapeptide sequence Trp-Met-Asp-PheNH2 or derivatives thereof, were studied. They were radioiodinated by the Iodogen or Bolton-Hunter procedures. The peptides tested were members of the gastrin- or cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety (occurring in native or sulfated form). Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing s.c. human MTC xenografts. Diethylene-triamine-pentaacetate derivatives of suitable peptides were synthesized, evaluated, and labeled with (111)In. All members of the CCK or gastrin family were stable in serum (with t(1/2)s of several hours at 37 degrees C); nevertheless, the stability of those peptides was highest that bore the NH2-terminal pGlu residues (e.g., big gastrin, gastrin-I, caerulein, and others) or D-amino acids. In accordance to their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues (e.g., the stomach). Sulfated CCK derivatives performed significantly better but additionally displayed a high uptake in normal, CCK-A receptor-expressing tissues (such as the liver/gallbladder, pancreas, and bowel). Best tumor uptake and tumor:nontumor ratios were obtained with members of the gastrin family, probably because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC bearing animals showed significant antitumor efficacy as compared with untreated controls. (111)In-Labeled diethylene-triamine-pentaacetate derivatives of minigastrin showed excellent targeting of CCK-B receptor-expressing tissues in animals and a normal human volunteer. These data suggest that CCK/gastrin analogues may be a useful new class of receptor binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, and hence, lower accretion in normal CCK-A receptor-expressing organs. Further preclinical as well as clinical studies are ongoing.

Intraduodenal fat inhibits gastric emptying and exerts early satiation in animals and humans, but it is not clear whether the effects are mediated by cholecystokinin (CCK) in humans. Here, we tested whether CCK-A receptors mediate the inhibition of fat on food intake. Two sequential, double-blind, crossover studies were performed in 24 male subjects. First, subjects received either intraduodenal fat or saline together with a preload of either water or banana shake. Second, 12 subjects received either intraduodenal fat or saline perfusion plus a concomitant infusion of saline or loxiglumide, a specific CCK-A receptor antagonist, together with a preload of banana shake. In both studies, subjects were free to eat and drink as much as they wished. Fat induced a reduction in calorie intake (P < 0.05) compared with controls. Furthermore, a decrease in hunger feelings was observed. Infusion of loxiglumide abolished the effects of fat. Duodenal fat interacts with an appetizer to modulate energy intake in humans. This effect is mediated by CCK-A receptors.

In this study we identified and characterized the receptor related to the modulation of growth of human gastric cancer by gastrin. By performing receptor binding assays on human AGS gastric cancer cells with the selective CCK-B/gastrin receptor antagonist [3H]L-365,260, specific and saturable binding were determined. Binding was dependent on protein concentration, time, temperature, and the presence of protease inhibitors, and was located in the membrane fraction. Gastrin, as well as CCK, stimulated gastric cancer cell growth in a receptor-mediated fashion at a concentration consistent with the binding affinity. Receptor gene expression for the CCK-B/gastrin receptor, but not for the CCK-A receptor, was found by reverse transcription polymerase chain reaction. Receptor binding assays as well as transcriptional and growth studies provide evidence that gastrin-stimulated growth of human gastric cancer is mediated by CCK-B/gastrin-like receptors.

Cholecystokinin (CCK)-A receptor (AR) and B receptor (BR) share highly amino acid sequence homology and overlap in their tissue distribution. We examined the anxiety-related behavior of CCK-AR, CCK-BR, and CCK-ARBR gene knockout (-/-) mice in the elevated plus-maze. CCK-AR(-/-) mice showed a significantly higher frequency of open-arm entries than wild-type and CCK-BR(-/-) mice, whereas the percentage open-arm entry values in CCK-AR(-/-) mice did not differ from those in wild-type mice. Thus, this increased frequency in open-arm entries for CCK-AR(-/-) mice was interpreted to be due to an increase in locomotor activity, rather than to a reduction in anxiety. By contrast, CCK-BR(-/-) mice showed significantly lower percentage open-arm entry values and spent significantly less time in the open- arms than wild-type and CCK-AR(-/-) mice. We therefore conclude that a lack of CCK-BR increases the anxiety-related behavior of the mouse in the elevated plus- maze.

To investigate the relative participation of peripheral (CCK-A) and central (CCK-B) cholecystokinin (CCK) receptors in satiety induced by endogenous CCK, we examined the effect of the CCK-A antagonist MK-329 (10-315 micrograms/kg) and the CCK-B antagonist L 365260 (0.1-315 micrograms/kg) on intake of a 20% sucrose solution in mildly food-deprived mice. Intraperitoneal injection of MK-329 elicited a dose-related increase in sucrose consumption with a minimal effective dose of 31.5 micrograms/kg. This dose increased sucrose intake 23% and the highest dose tested, 315 micrograms/kg, increased sucrose intake 63% above baseline. In contrast to MK-329, intraperitoneal administration of L 365260 had no effect on sucrose intake at doses up to 315 micrograms/kg. To examine the contribution of these two CCK receptor subtypes in satiety induced by exogenous CCK, CCK-8 (8 micrograms/kg) was administered alone and in combination with MK-329 and L 365260. MK-329 (10 micrograms/kg) significantly attenuated the satiety effect of CCK-8, and L 365260 (100 micrograms/kg) was without effect. These results suggest that the peripheral CCK receptor subtype mediates satiety induced by endogenous and exogenous CCK in the mouse.

1. Morphine inhibits supraoptic nucleus oxytocin neurones directly and presynaptically via inhibition of afferent noradrenergic endings. 2. We studied whether morphine tolerance/dependence (induced by intracerebroventricular (I.C.V.) morphine infusion) alters the responsiveness of oxytocin neurones to systemic cholecystokinin (CCK), a stimulus which activates oxytocin neurones via the release of noradrenaline. 3. CCK (20 micrograms kg-1, i.v.) increased plasma oxytocin concentrations similarly in urethane-anaesthetized morphine-naive and -dependent rats. In naive rats, I.C.V. (10 micrograms) and i.v. morphine (0.5 mg kg-1) reduced CCK-induced oxytocin secretion by 95 +/- 4 and 49 +/- 10%, respectively. In dependent rats, i.v. morphine reduced CCK-induced release by only 8 +/- 9%, indicating tolerance. 4. In urethane-anaesthetized rats, i.v. CCK increased the firing rates of oxytocin neurones similarly in morphine-naive and -dependent rats (by 1.2 +/- 0.2 and 1.4 +/- 0.3 spikes s-1 maximum, respectively, over 5 min). Naloxone did not alter spontaneous or CCK-induced activity in naive rats but increased activity in dependent rats (by 3.4 +/- 0.5 spikes s-1), indicative of withdrawal excitation; however, the response to CCK remained unchanged after naloxone. 5. Systemic CCK did not trigger withdrawal, nor did it have a greater excitatory effect in dependent rats. Thus, morphine withdrawal excitation of oxytocin neurones does not involve supersensitivity to the noradrenergic input, or hypersensitivity of this input to i.v. CCK. Tolerance apparently occurs both at the cell bodies of oxytocin neurones in the supraoptic nucleus and in their noradrenergic input. However, dependence is apparent only at the cell bodies.

Regulatory peptides, such as vasoactive intestinal peptide (VIP), somatostatin (SS), or substance P (SP), are considered to play a role in immune regulation. To localize the targets of these peptides in the human immune system, their receptors have been evaluated with in vitro receptor autoradiography in lymph nodes, tonsils, appendix, Peyer's patches, spleen, and thymus. The three peptide receptors were detected in all lymphoid tissues tested, but, unexpectedly, usually in distinct compartments. In lymph nodes, palatine tonsils, vermiform appendix, and Peyer's patches, VIP receptors were found in the CD3 positive zone around lymphoid follicles; SS receptors in the germinal centers of secondary follicles; and SP receptors mainly in interfollicular blood vessels. In the spleen, VIP receptors were detected in periarterial lymphatic sheaths, SS receptors in the red pulp, and SP receptors in the central arteries. In the thymus, VIP receptors were present in cortex and medulla, SS receptors in the medulla, and SP receptors in blood vessels. For comparison, cholecystokinin (CCK)-A and -B receptors were not demonstrated in any of these tissues. These results suggest a strong compartmentalization of the three peptide receptors in human lymphoid tissues and represent the molecular basis for the understanding of a very complex and interactive mode of action of these peptides.

An immunocytochemical method was used for localization of various peptide-like substances in the Ascaris nervous system. Out of 45 antipeptide antisera, 12 demonstrated immunoreactivity in different subsets of neurons; these 12 antisera were raised against luteinizing hormone-releasing hormone (LHRH), Aplysia peptide L11 (L11), Aplysia peptide 12B (12B), small cardioactive peptide B (SCPB), neuropeptide Y (NPY), FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), alpha-melanocyte stimulating hormone (alpha MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF), and vasoactive intestinal peptide (VIP). Several peptide-like substances were colocalized to the same neuron. Our results suggest that Ascaris, like other organisms, contains multiple peptidergic systems.

The corticotrophin-releasing factor (CRF) is shown to be released during stress suggesting that CRF has a physiological role in the mediation of central nervous system (CNS) response to stress, including an inhibitory effect on gastric emptying. In the present study, we have examined the pathways by which intracerebroventricularly (i.c.v.) administered CRF and central CRF activation during stress alter the gastric emptying rate of saline (0.14 M), acid (50 mM), peptone (4.5%) and peptone after preload. The emptying rates of all these test meals were significantly (p < 0.05-0.001) delayed with increasing doses of i.c.v. CRF (0.001, 0.003, 0.01, 0.1, 0.3 and 1 nmol/10 microl), when compared with their i.c.v. saline-treated controls. The 1-nmol dose of CRF inhibited the emptying of acid, peptone and peptone after a preload by 43.8%, 64.1% and 81.1%, respectively. Twenty-minute swim stress delayed gastric emptying rate of saline, acid and peptone solutions significantly (p < 0.001) and the CRF receptor antagonist, alpha-helical CRF (8 nmol/10 microl, i.c.v.), applied before the swim stress, abolished the inhibitory effect of stress on the emptying rate of these solutions. Acute intragastric administration of capsaicin (2 mg/rat) and systemic capsaicin (125 mg kg(-1)) treatment facilitated the gastric emptying rate of acid, peptone and peptone after preload significantly, almost abolishing the inhibitory effect of central CRF (p < 0.001). However, either capsaicin treatment had no effect on stress-induced inhibition of the gastric emptying of none of the solutions, except peptone after a preload. Our findings demonstrate that the gastric inhibitory response induced by swimming as a stress-producing stimulus is mediated by the endogenous release of CRF. They also suggest that CRF exerts its CNS actions on the gastrointestinal tract via vago-vagal, capsaicin-sensitive pathways, probably involving the central cholecystokinin (CCK) mechanisms.

The transcription factor NF-kappaB (nuclear factor kappaB) is a central mediator of inflammatory and apoptotic signaling in the cell. The protein kinase RIP-2 is a member of the CARD protein family (caspase activation and recruitment domain, also known as CARD3, Ripk2, CARDIAK, RICK, and CCK), and has been shown to be an activator of NF-kappaB. In this study, it was demonstrated by transcriptional profiling and protein expression analysis that the inflammatory cytokines TNF-alpha, IL-1 beta, and IFN-gamma induced RIP-2 transcription and translation in endothelial cells. Two mechanistically distinct inhibitors of NF-kappaB signaling, sulfasalazine (NF-kappaB inhibitor) and WY-14643 [PPAR alpha (peroxisome proliferator-activated receptor alpha) agonist] that interfere with the transcription factor RELA (p65), suppressed TNF-alpha induced RIP-2 gene expression, which indicated that NF-kappaB signaling was involved in the cytokine-induced transcriptional activation of RIP-2 gene expression. Consistent with these observations, multiple NF-kappaB response elements were found in the upstream regions of the human and mouse RIP-2 genes. NF-kappaB-mediated regulation of RIP-2 gene and protein expression suggests an additional step in the regulation of NF-kappaB function as RIP-2 has been shown to positively modulate NF-kappaB by binding IKK gamma (I kappaB kinase gamma), a component of the IKK complex. These findings support a positive feed-forward mechanism of NF-kappaB regulation that involves NF-kappaB-dependent induction of RIP-2 transcription and a subsequent increase in RIP-2 protein levels in response to inflammatory cytokines. Elevated RIP-2 protein levels are then available to promote NF-kappaB function via interaction with IKK gamma. RIP-2 is the first reported NF-kappaB-dependent protein kinase that positively regulates NF-kappaB activity.

In this study, a PstI polymorphic site with two individual alleles, namely AACCK) type A receptor gene. The PstI polymorphic site was used as a genetic marker to study its association with psychotic symptoms in schizophrenia. A significant difference in allelic frequency was found between schizophrenic patients with and without auditory hallucinations(chi(2) = 6.26, df = 1, P = 0.012), and the odds ratio for the allelic association was 2.21 (95% CI 1.18-4.15) with an attributable fraction of 0.1. The frequency of AAAACCK-A receptor gene may be associated with auditory hallucinations in schizophrenia.

Inhibition of colon carcinoma cell growth by the nonselective gastrin/cholecystokinin (CCK) receptor antagonists proglumide and benzotript provided evidence that gastrin functions as an autocrine growth factor. However, the molecular properties of the receptor mediating the antagonist effects have not been identified. A 78-kDa gastrin-binding protein (GBP), the sequence of which is related to the family of enzymes possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, has been previously purified from porcine gastric mucosal membranes. I now report that covalent cross-linking of 125I-labeled [Nle15]gastrin2,17 to the 78-kDa GBP is inhibited by crotonyl-CoA and by acetoacetyl-CoA. Gastrin, CCK, and their analogues also inhibit cross-linking, and the spectrum of analogue affinities correlates better with the values previously reported for binding to the gastrin/CCK-C receptor than with the values reported for binding to either the CCK-A or the gastrin/CCK-B receptor. Cross-linking is also inhibited by proglumide and benzotript, but no inhibition is seen with either the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B receptor-selective antagonist L365,260. The affinities of antagonists for the GBP correlate well with their affinities for the gastrin/CCK-C receptor and with their potencies for inhibition of colon carcinoma cell growth. I conclude that the 78-kDa gastrin-binding protein is (i) a member of the hydratase/dehydrogenase family of fatty acid oxidation enzymes, (ii) the gastrin/CCK-C receptor, and (iii) the target for the antiproliferative action of two gastrin/CCK receptor antagonists.

OBJECTIVE

To elucidate the anti-inflammatory mechanism of an intestinal neuropeptide, sulfated cholecystokinin octapeptide (sCCK-8), the effects of sCCK-8 on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) mRNA expression and NF-kappaB activity in pulmonary interstitial macrophages (PIMs) were studied.

METHODS

PIMs from rat were stimulated with LPS (1 mg.L(-1)) in the presence or absence of sCCK-8 (10(-8)-10(-6)mol.L(-1)) or/and CCK receptor antagonist proglumide (2 mg.L(-1)). The expression of TNF-alpha mRNA was assayed by reverse transcription polymerase chain reaction (RT-PCR) at 3h of the stimulation, and nuclear factor-kappaB (NF-kappaB) binding activity was analyzed by electrophoretic mobility shift assay (EMSA) at 1 h of stimulation. The IkappaBalpha protein level in the cytoplasma at 30 min of the stimulation was detected by Western blot.

RESULTS

sCCK-8, at concentrations from 10(-8) mol.L(-1) to 10(-6) mol.L(-1) obviously inhibited LPS-induced TNF-alpha mRNA expression and NF-kappaB binding activity in a dose-dependent manner, P<0.05, P<0.01. Stimulation PIMs with LPS resulted in a reduction of IkappaBalpha protein level, P<0.01, which was elevated by sCCK-8, P<0.05. The effects of sCCK-8 on NF-kappaB activity and IkappaB protein level were attenuated by CCK receptor antagonist proglumide, P<0.01.

CONCLUSIONS

sCCK-8 inhibits LPS-induced TNF-alpha mRNA expression by regulating NF-kappaB activity in rat PIMs, which is mediated through CCK receptors and inhibiting IkappaB-alpha degradation. This represents one of the anti-inflammatory mechanisms of sCCK-8.

Feeding induces increased sleep in several species, including rats. The aim of the study was to determine if CCK plays a role in sleep responses to feeding. We induced excess eating in rats by 4 days of starvation and studied the sleep responses to refeeding in control and CCK-A receptor antagonist-treated animals. Sleep was recorded on 2 baseline days when food was provided ad libitum. After the starvation period, sleep was recorded on 2 refeeding days when the control rats (n = 8) were injected with vehicle and the experimental animals (n = 8) received intraperitoneal injections of L-364,718 (500 microg/kg, on both refeeding days). In the control group, refeeding caused increases in rapid eye movement sleep (REMS) and non-REMS (NREMS) and decreases in NREMS intensity as indicated by the slow-wave activity (SWA) of the electroencephalogram. CCK-A receptor antagonist treatment completely prevented the SWA responses and delayed the NREMS responses to refeeding; REMS responses were not simply abolished, but the amount of REMS was below baseline after the antagonist treatment. These results suggest that endogenous CCK, acting on CCK-A receptors, may play a key role in eliciting postprandial sleep.

Duodenal fat such as oleate is known to influence gut functions by release of cholecystokinin (CCK), but the contribution of CCK endogenously released by duodenal fat or by diversion of pancreatic juice from the duodenum in the mechanism of mucosal integrity and gastroprotection has been little studied. This study was designed to compare the effect of CCK-8 and intraduodenal (i.d.) instillation of sodium oleate, or diversion of the pancreatic biliary secretions that are known to release CCK, on the gastric mucosal lesions induced by topical application of 100% ethanol or acidified aspirin (ASA) in rats with or without the pretreatment with a CCK-A receptor antagonist, loxiglumide, or with L-365,260 to block CCK-B receptors. In addition, the effect of suppression of prostaglandin (PG) biosynthesis by indomethacin (5 mg/kg i.p.), inhibition of nitric oxide (NO)-synthase by L-NAME (5 mg/kg i.v.), or blockade of sensory nerves by capsaicin (125 mg/kg s.c.) on the protective activity of sodium oleate was determined. Sodium oleate (50-200 mM i.d.), or diversion of pancreatic juice from the duodenum for 3 h that produced significant rise in plasma CCK levels, significantly reduced gastric lesions induced by 100% ethanol to an extent similar to that induced by exogenous CCK-8 (5 nmol/kg s.c.). The protective effect of oleate or diversion of pancreatic juice was accompanied by an increase in gastric blood flow (GBF). Both protection and accompanying hyperemia were completely abolished by blockade of CCK-A receptors with loxiglumide, whereas L-365,260, an antagonist of CCK-B receptors, had no effect. Oleate given i.d. significantly attenuated acidified ASA-induced gastric lesions and gastric secretion while increasing the luminal concentration of somatostatin. These effects were significantly reduced by loxiglumide but not by L-365,260. In contrast, CCK-8, which stimulated gastric acid secretion, failed to affect the lesions induced by acidified ASA and the decrease in the GBF produced by this ulcerogen. Indomethacin, which suppressed PG generation by approximately 90%, failed to influence the protective activity of oleate or CCK-8 against ethanol-induced lesions, whereas L-NAME, vagotomy, or sensory denervation significantly attenuated this protection and accompanying hyperemia. Addition to L-NAME of L-arginine, but not D-arginine, restored the protective and hyperemic effects of CCK-8 and duodenal oleate against gastric lesions induced by ethanol or acidified ASA. We conclude that endogenous CCK released by oleate or diversion of pancreatic secretion exerts a potent gastroprotective action on the stomach involving predominantly CCK-A receptors and depending on vagal activity, and hyperemia mediated by NO and sensory nerves but unrelated to acid secretory effects and endogenous PG.