A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, alpha-endorphin, beta-endorphin, gamma 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before. The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to beta-endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.

Despite the extensive amino acid homology between gastrin and cholecystokinin (CCK) at the biologically active carboxyl terminus, the receptors through which these peptides exert their action are heterogeneous. In previous studies, we have examined the biological activity of gastrin/CCK peptides on isolated canine fundic D-cells and observed that CCK is a more potent and efficacious stimulant of somatostatin release than gastrin. We performed the present studies to distinguish between distinct CCK (CCK-A subtype) and gastrin (CCK-B/gastrin subtype) receptors on canine D-cells. Consistent with this observation was our finding that the CCK-A receptor selective antagonist L-364,718 dose dependently (10(-11)-10(-7) M) inhibited CCK-mediated somatostatin release but at the same doses did not alter the effect of gastrin. CCK and gastrin exhibited similar potency in displacing bound 125I-labeled Leu15 gastrin-17 from D-cells. However, when 125I-CCK octapeptide (CCK-8) was used as the radioligand, a fraction of the bound label could not be displaced with gastrin, but this fraction was completely displaced with CCK-8. In D-cells pretreated with high concentrations of gastrin, L-364,718 was able to inhibit the gastrin-resistant fraction of 125I-CCK-8 binding, but the CCK-B/gastrin receptor selective antagonist (PD 134308) was unable to influence this fraction of binding in doses as high as 10(-6) M. These studies delineate the presence of distinct CCK-A and CCK-B/gastrin receptors on canine fundic D-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The brain-gut hormones, cholecystokinin (CCK) and gastrin, regulate the growth of gastrointestinal mucosa and tumor cells. In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate messenger RNA expression for CCK, gastrin, CCK-A receptor, and CCK-B/gastrin receptor in surgical specimens of gastric cancers and in normal antrum and body mucosa of the stomach. The CCK mRNA expression was detectable in 4/14 (29%) samples of gastric cancer and in 3/12 (25%) samples of antral mucosa. However, the gastrin mRNA expression was not detectable in any gastric cancer samples, although it was detectable in all the samples of antral mucosa. The CCK-A receptor mRNA expression was detectable in 5/14 (36%) samples of gastric cancer and in 7/12 (58%) body mucosa. Three cases out of 14 (21%) of gastric cancer expressed both CCK gene and CCK-A receptor gene. The CCK-B receptor mRNA expression was detectable in only 1/14 (7%) samples of gastric cancer, although it was detectable in 10/12 (83%) body mucosa of the stomach. These findings may suggest a greater role for CCK and CCK-A receptor than for gastrin and CCK-B receptor in gastric cancers.

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in rats.

The purpose of this study is to determine whether the level of cholecystokinin (CCK) receptor expression causes the differences between the responder and non-responder to electroacupuncture mediated analgesic effects. Male Sprague-Dawley rats were stimulated at the Zusanli (ST36) acupoint in the absence of any anesthetics and holders. The tail flick latency test was performed to quantify analgesic effects and then the responder and non-responder groups were classified. The hypothalamus of each group was dissected and RNA was purified. The amount of mRNA expression of CCK-A and CCK-B receptors was determined by reverse transcription-polymerase chain reaction. The results show that CCK-A receptors are significantly more expressed in non-responders than responders, whereas CCK-B receptor expression is similar in both groups.

The sympathetic nervous system is known to innervate the pancreatic islets and to have the capability to influence islet hormone release. The effects are, however, complex since the islet nerves contain catecholaminergic as well as peptidergic fibres, and the catecholamines stimulate alpha- as well as beta-adrenoceptors. The present study was undertaken to establish the possible influence of the alpha-adrenoceptors on basal and stimulated insulin secretion under in vivo conditions. The alpha-adrenoceptor blocker phentolamine was injected at various dose levels i.p. to mice and a dose-dependent increase in plasma concentrations of insulin was seen. The maximal plasma insulin levels were observed 10 min after injection and were accompanied by decreased plasma glucose concentrations. Additionally, plasma glucose levels fell in response to phentolamine by an apparent insulin-independent manner, since at the low dose of 2.6 mumol kg-1, plasma glucose levels did decrease without any apparent increase in plasma insulin levels. After injection of a low dose of phentolamine 10 min prior to a rapid i.v. injection of one of four different insulin secretagogues, the following effects on insulin release were observed. Glucose (+55%) and the cholinergic agonist carbachol (+140%) displayed a potentiated insulin secretory response after phentolamine pretreatment, whereas the beta 2-adrenoceptor agonist terbutaline (-45%) had a blunted, though not abolished, insulin response. The absolute insulin secretory response to CCK-8 was unaffected by phentolamine despite the fact that plasma glucose levels were lowered by phentolamine. In conclusion, phentolamine enhanced insulin secretion and depressed plasma glucose levels in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The etiology of gallstones is multifactorial, with interactions between genes and the environment. We generated cholecystokinin (CCK) -A receptor (R)-deficient (-/-) mice and found that CCK did not produce gallbladder contraction in CCK-AR(-/-) mice. The purpose of this study was to identify the role of CCK-AR on gallstone formation. Age-matched CCK-AR gene (+/+) and (-/-) progenies were used. Sludge and gallstone formation, as well as plasma cholesterol levels, were measured at 12 and 24 months of age. Sludge and gallstone formation were significantly higher in CCK-AR(-/-) mice than in CCK-AR(+/+) mice at 12 and 24 months of age, although these were not different between 12 and 24 months of age. The plasma cholesterol levels, daily food intake, and body weight were not significantly different between CCK-AR(+/+) and (-/-) mice. Sludge and gallstone formation were not observed at 6 months of age. In conclusion, deteriorated gallbladder contraction due to a lack of CCK-AR favored gallstone formation after the middle age of life.

Functional dyspepsia (FD) is a common disorder of yet uncertain etiology. Dyspeptic symptoms are usually meal related and suggest an association to gastrointestinal (GI) sensorimotor dysfunction. Cholecystokinin (CCK) is an established brain-gut peptide that plays an important regulatory role in gastrointestinal function. It inhibits gastric motility and emptying via a capsaicin sensitive vagal pathway. The effects on emptying are via its action on the proximal stomach and pylorus. CCK is also involved in the regulation of food intake. It is released in the gut in response to a meal and acts via vagal afferents to induce satiety. Furthermore CCK has also been shown to be involved in the pathogenesis of panic disorder, anxiety and pain. Other neurotransmitters such as serotonin and noradrenaline may be implicated with CCK in the coordination of GI activity. In addition, intravenous administration of CCK has been observed to reproduce the symptoms in FD and this effect can be blocked both by atropine and loxiglumide (CCK-A antagonist). It is possible that an altered response to CCK may be responsible for the commonly observed gastric sensorimotor dysfunction, which may then be associated with the genesis of dyspeptic symptoms.

In rodents, cholecystokinin (CCK) induces pancreatic enzyme secretion and pancreas growth through its CCK(A) receptors. It is unknown whether occupation of the CCK(B) receptors present in pig and human pancreas can cause the same effects. This study evaluates CCK(B) receptor expression in rat, mouse, pig, and fetal human pancreata using Northern blot, Western blot, and immunofluorescence techniques. The reported 2.7-kb CCK(B) receptor mRNA transcript in the rat brain and gastric fundus is absent in pancreas; the message was, however, detected by RT-PCR and by a CCK(B) receptor antibody as an 80-kDa protein present uniquely in islet delta-cells. Proteins of 50 and 80 kDa appear in mouse pancreas, and proteins of 50 and 115 kDa appear in pig and human pancreas, respectively, all localized in islet delta-cells. Gastrin mRNAs are strongly present in fetal rat pancreas, and the hormone is localized in islets; both are repressed 10 days after birth. In conclusion, the CCK(B) receptors are present in pancreas of four species with exclusive location in islet delta-cells. In such a location, they could be indirectly involved in the control of enzyme secretion.

Heat shock proteins (HSPs) are cytoprotective proteins that are expressed constitutively and/or at elevated levels upon the exposure of cells to stress. The aim of this study was to investigate the potential effects of HSP preinduction by cold- (CWI) or hot-water immersion (HWI) on pro-inflammatory cytokine production (IL-1, IL-6, TNF-alpha) in cholecystokinin-octapeptide(CCK)-induced acute pancreatitis. Rats were injected with 3 x 75 microg/kg CCK subcutaneously at intervals of 2 h at the peak level of HSP synthesis, as determined by Western blot analysis. The animals were killed by exsanguination through the abdominal aorta 2 h after the last CCK injection. The serum IL-1, IL-6, TNF-alpha, and amylase levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase and trypsinogen were measured; biopsy for histology was taken. HWI significantly elevated the HSP72 expression, while CWI significantly increased the HSP60 expression. HWI pretreatment decreased all of the measured serum cytokine levels in this acute pancreatitis model. CWI and HWI pretreatment ameliorated most of the examined laboratory and morphological parameters of CCK-induced pancreatitis. The findings suggest the possible roles of HSP60 and HSP72 in the protection against CCK-induced pancreatitis. HSP72 might also participate in the reduction of pro-inflammatory cytokine synthesis.

A comparative map of human chromosome 3 (HSA 3) and pig chromosome 13 (SSC 13) was constructed using physically assigned pig sequence-tagged sites (STSs). Pig STSs representing 11 HSA 3 genes, including v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF1), retinoic acid beta receptor (RARB), cholecystokinin (CCK), pituitary transcription factor 1 (POU1F1), ceruloplasmin (CP), guanine nucleotide binding protein, alpha-inhibiting polypeptide 2 (GNAI2), sucrase-isomaltase (SI), rhodopsin (RHO), dopamine receptor D3 (DRD3), growth-associated protein 43 (GAP43), and somatostatin (SST), were developed. Ten pig STSs were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC 13 with 80-100% concordance. Large-insert probes were obtained by screening a pig yeast artificial chromosome (YAC) library with primers for each STS. Several YACs were identified for DRD3, GAP43, POU1F1, RHO, SI, and SST for fluorescence in situ hybridization (FISH) mapping. Single gene and bi-color FISH with each pairwise combination were used to further define the gene order on SSC 13. While these data confirm chromosome painting results showing that HSA 3 probes hybridize to a major portion of SSC 13, they also demonstrate extensive gene-order differences between man and pig within this large conserved synteny group. Interestingly, several conserved chromosomal regions have been detected between pig and mouse that are not conserved between man and mouse, suggesting that the SSC 13 gene arrangement may be the closest to that of the ancestral eutherian chromosome.

Cholecystokinin (CCK) is a known stimulus for the release of insulin and other islet hormones. To localize islet cell CCK binding sites, we measured the uptake of 125I-CCK by the isolated, perfused rat pancreas. Light microscope autoradiographs revealed uptake of label over both the endocrine islets of Langerhans and the exocrine acini. This uptake of 125I-CCK was saturable, as it decreased markedly when a large excess of unlabeled CCKCCK, electron microscope autoradiographs were prepared. The majority of silver grains in islets were localized over beta cells (69%), although saturable uptake was also observed over alpha (12%) and other islet cells. When grain densities were analyzed (grains/micron 2), the highest density was observed over islet blood vessel cells. In contrast to islet blood vessels, there was no localization of 125I-CCK over acinar blood vessels. This study supports the concept, therefore, that there is a direct regulation of islet endocrine cells by CCK, and also raises the possibility that CCK influences islet hormone release via an indirect effect on the islet vascular endothelium.

In the present study, we investigated whether cholecystokinin (CCK) or its structurally related peptide gastrin participates in long term regulation of adipocyte leptin secretion. The levels of circulating leptin observed after 2 and 6 h of refeeding in 18-h fast rats were significantly lowered by injection of the specific gastrin/CCK-B receptor antagonist YM022 at doses that did not affect feeding behavior. Moreover, in normally fed animals, circulating leptin was markedly decreased by chronic injection of YM022 (from 4 +/- 0.6 to 2.1 +/- 0.5 ng/ml). Consistent with these observations, YM022 treatment decreased leptin messenger RNA (mRNA) levels and increased the leptin content in rat epididymal fat tissue. Rat adipocytes exclusively contain gastrin/CCK-B receptor mRNA, but not CCK-A receptor mRNA. Furthermore, adipocyte membranes bound [125I]CCK-8 in a saturable manner, with kinetics consistent with a single class of high affinity sites with a Kd of 0.2 nM. These data argue for a physiological role for the CCK-B/gastrin receptor in adipocyte leptin regulation. We therefore propose that gastrin is involved in long term regulation of leptin expression and secretion in rat fat tissues through activation of an adipocyte gastrin/CCK-B receptor.

Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition.

Eating disorders (EDs) have a highly heterogeneous etiology and multiple genetic factors might contribute to their pathogenesis. Ghrelin, a novel growth hormone-releasing peptide, enhances appetite and increases food intake, and human ghrelin plasma levels are inversely correlated with body mass index. In the present study, we examined the 171T/C polymorphism of the ghrelin receptor (growth hormone secretagogue receptor, GHSR) gene in patients diagnosed with EDs, because the subjects having ghrelin gene polymorphism (Leu72Met) was not detected in a Japanese population, previously. In addition, beta3 adrenergic receptor gene polymorphism (Try64Arg) and cholecystokinin (CCK)-A receptor (R) gene polymorphism (-81A/G, -128G/T), which are both associated with obesity, were investigated. The subjects consisted of 228 Japanese patients with EDs [96 anorexia nervosa (AN), 116 bulimia nervosa (BN) and 16 not otherwise specified (NOS)]. The age- and gender-matched control group consisted of 284 unrelated Japanese subjects. The frequency of the CC type of the GHSR gene was significantly higher in BN subjects than in control subjects (chi(2) = 4.47, p = 0.035, odds ratio = 2.05, Bonferroni correction: p = 0.070), while the frequency in AN subjects was not different from that in controls. The distribution of neither beta3 adrenergic receptor gene nor CCK-AR polymorphism differed between EDs and control subjects. Therefore, the CC type of GHSR gene polymorphism (171T/C) is a risk factor for BN, but not for AN.

PKC-delta is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-delta TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-delta TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-delta TYR-P by activation of the low affinity CCK(A) receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-delta TYR-P but instead, increased it. ACCK-stimulated PKC-delta TYR-P. GF 109203X, at low concentrations, increased PKC-delta TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, Gö 6976 and safingol, caused a similar increase in TPA- and CCK-stimulated PKC-delta TYR-P. These results demonstrate that CCK(A) receptor activation causes PKC-delta TYR-P through activation of only one of its two receptor affinity states. This PKC-delta TYR-P is not directly influenced by changes in [Ca(2+)](i); however, the resultant activation of PKC-alpha has an inhibitory effect. Therefore, CCK activates both stimulatory and inhibitory PKC cascades regulating PKC-delta TYR-P and, hence, likely plays an important role in regulating PKC-delta degradation and cellular abundance.

Cholecystokinin (CCK) is a classical brain-gut peptide that exerts a variety of physiological actions in the gastrointestinal tract and central nervous system. CCK occurs in several molecular forms of varying aminoacid length, the sulphated octapeptide (CCK-8) being the predominant form in the brain. CCK mediates its effects through interaction with specific receptors subdivided in two subtypes--CCK-A (present in the periphery and in few selected brain nuclei) and CCK-B (the predominant receptor subtype in the brain). CCK is implicated in variety of behavioral functions as satiety, anxiety, exploratory and locomotor activity and learning and memory. After a brief description of the distribution, molecular forms, release, inactivation, etc. of CCK in the brain, the present review summarizes the recent data on the role of CCK in learning and memory. The memory-enhancing effects of CCK have been demonstrated in various types of memory. Data showing that CCK-A receptors mediate mnemonic while CCK-B receptors mediate amnestic effects are also presented.

We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM-10 nM) and carbachol (0.1-100 microM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 microM) and the phospholipase C (PLC) inhibitor U-73122 (10 microM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 microM). The pp60(c-src) inhibitor herbimycin A (6 microM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 microM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 microM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.

The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated G(q/11), G(13), and G(12), and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G(13) and inhibited by Galpha(13) antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 +/- 2% to 53 +/- 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Galpha(13) antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-alpha inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G(12) was not coupled to RhoA or ARF, and Galpha(12) antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G(13)-dependent RhoA and by ARF and PKC-alpha and is modulated by an inhibitory G(12)-dependent pathway.

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.

"Responder" Sprague-Dawley (SD) rats that were sensitive to electroacupuncture (EA) in an acute thermal pain test (i.e. tail flick latency [TFL] test) maintained sensitivity to EA in the warm allodynia test after peripheral nerve injury. Similarly, the "non-responder" SD rats that were insensitive to EA in the TFL test were also insensitive to EA in the allodynia test. The EA-induced analgesic effects in the TFL test were significantly higher in CCK-A receptor deficient, Otsuka Long-Evans Tokushima Fatty (OLETF) rats than in their littermates, Long-Evans Tokushima Otsuka (LETO) rats. Similarly, the anti-allodynic effects of EA were significantly greater in OLETF rats than in LETO rats. These results suggest that the individual differences in the sensitivity of acute pain behavior to EA were maintained in neuropathic pain behavior following peripheral nerve injury, and that CCK-A receptor expression plays an important role in mediating this phenomenon.

The cholecystokinin (CCK) receptor involved in contraction of guinea pig ileal longitudinal muscle to cholecystokinin is poorly understood; some studies have suggested that contraction was mediated via a CCK-A receptor whereas other studies have implicated CCK-B receptors in ileal contraction to CCK. To clarify this, we compared the effects of CCK-8 sulfate, CCK-4 and gastrin in radioligand binding studies and longitudinal ileal contractility in vitro. Contraction to all three peptides was abolished by tetrodotoxin (3 x 10(-7)M), confirming the neuronal nature of the CCK receptors mediating contraction to all three peptides. Maximal CCK-8S contractions were inhibited by 80% in the presence of atropine (10(-6)M), and entirely by the combination of atropine and a substance P receptor antagonist (3 x 10(-5)M). CCK-4 and gastrin-induced contractions were unaffected by substance P receptor blockade, but were abolished by atropine. Two selective CCK-A and CCK-B receptor antagonists, L-364,718 and L-365,260, respectively, were used to probe further the receptors involved in ileal contraction to this peptide family. Radioligand binding studies in mouse brain, rat pancreas and guinea pig stomach confirmed the selectivity of these antagonists. The CCK-A selective antagonist, L-364,718, potently inhibited ileal contractions to CCK-8S (-log KB = 9.35) with 10-fold lower affinity at receptors mediating contraction to CCK-4 (-log KB = 8.25). In contrast, the CCK-B receptor antagonist, L-365,260, did not affect contraction to CCK-8S (-log KB less than 7) but potently inhibited contraction to CCK-4 (-log KB = 9.24).(ABSTRACT TRUNCATED AT 250 WORDS)

The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCK(A) receptor (AdCCK(A)R) was used to express the rat receptor in acini from CCK(A) receptor-deficient mice (CCK(A)R -/-). Infection of CCK(A)R -/- acini in vitro with pAdCCK(A)R led to a time-dependent increase in (125)I-CCK(8) binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCK(A) receptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCK(A)R JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCK(A)R -/- mouse acini infected with AdCCK(A)R resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCK(A) receptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180.