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In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm−2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications.

Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.

Background Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the μ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed. Results Forty-eight hours after treating SH-SY5Y cells with morphine (10 μM), naloxone (10 μM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1–10 μM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 μM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1–10 μM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor. Conclusions This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

Cholecystokinin (CCK) is a brain‐gut peptide; it functions both as a neuropeptide and as a gut hormone. Although the pancreas and the gallbladder were long thought to be the principal peripheral targets of CCK, CCK receptors are found throughout the gut. It is likely that CCK has a physiological role not only in the stimulation of pancreatic and biliary secretions but also in the regulation of gastrointestinal motility. The motor effects of CCK include postprandial inhibition of gastric emptying and inhibition of colonic transit. It is now evident that at least two different receptors, CCK1 and CCK2 (formerly CCK‐A and CCK‐B, respectively), mediate the actions of CCK. Both localization and functional studies suggest that the motor effects of CCK are mediated by CCK1 receptors in humans. Since CCK is involved in sensory and motor responses to distension in the intestinal tract, it may contribute to the symptoms of constipation, bloating and abdominal pain that are often characteristic of functional gastrointestinal disorders in general and irritable bowel syndrome (IBS), in particular. CCK1 receptor antagonists are therefore currently under development for the treatment of constipation‐predominant IBS. Clinical studies suggest that CCK1 receptor antagonists are effective facilitators of gastric emptying and inhibitors of gallbladder contraction and can accelerate colonic transit time in healthy volunteers and patients with IBS. These drugs are therefore potentially of great value in the treatment of motility disorders such as constipation and constipation‐predominant IBS. British Journal of Pharmacology (2004) 141, 1275–1284. doi:10.1038/sj.bjp.0705769

Background and purpose: Leptin regulates energy expenditure and body weight by acting both on the hypothalamus and on peripheral targets. Central actions of leptin are enhanced by cholecystokinin (CCK). The interaction between leptin and CCK makes physiological sense, as rats lacking CCK1 receptors are resistant to peripheral leptin but not to leptin directly infused into the brain. We have recently reported that CCK enhances leptin effects by increasing the entry of leptin into the CNS. The aim of this work was to further characterize the effect of CCK (10 μg kg−1) on leptin kinetics as well as the CCK receptor subtype involved in the interaction between CCK and leptin. Experimental approach: Experiments were carried out both in free‐feeding and in fasted rats receiving a single dose of leptin (100 μg kg−1; i.p.). Parameters analysed over the next 6 h were plasma and cerebrospinal fluid concentrations of leptin. Key results: We observed that CCK‐8 depressed the increase in plasma leptin that followed the i.p. injection and simultaneously increased leptin concentration in the cerebrospinal fluid from 92±25 to 230±24 pg mL−1 (P<0.05). The effect of CCK‐8 was totally prevented by the CCK1 receptor antagonist, SR‐27,897 (0.3 mg kg−1, s.c.), but not by the CCK2 receptor antagonist, L‐365,260 (1 mg kg−1). Conclusions and implications: These results show that CCK plays a role in regulating the access of leptin to the brain and suggest that CCK analogues, acting on CCK1 receptors, might be useful drugs in improving leptin actions within the brain. British Journal of Pharmacology (2008) 154, 1009–1015; doi:10.1038/bjp.2008.149; published online 21 April 2008

Background and purpose: Acute intraperitoneal (i.p.) administration of cholecystokinin (CCK) is known to induce a significant, but short‐lasting, reduction in food intake, followed by recovery within hours. Therefore, we had covalently coupled CCK to a 10 kDa polyethylene glycol and showed that this conjugate, PEG‐CCK9, produced a significantly longer anorectic effect than unmodified CCK9. The present study assessed the dose–dependency of this response and the effect of two selective CCK1 receptor antagonists, with different abilities to cross the blood‐brain barrier (BBB), on PEG‐CCK9‐induced anorexia. Experimental approach: Food intake was measured, for up to 23 h, after i.p. administration of different doses (2, 4, 8, 16 and 32 μg kg−1) of CCK9 or PEG‐CCK9 in male Wistar rats. Devazepide (100 μg kg−1), which penetrates the BBB or 2‐NAP (3 mg kg−1), which does not cross the BBB, were coadministered i.p. with PEG‐CCK9 (6 μg kg−1) and food intake was monitored. Key results: In PEG‐CCK9‐treated rats, a clear dose‐dependency was seen for both the duration and initial intensity of the anorexia whereas, for CCK9, only the initial intensity was dose‐dependent. Intraperitoneal administration of devazepide or 2‐NAP, injected immediately prior to PEG‐CCK9, completely abolished the anorectic effect of PEG‐CCK9. Conclusions and implications: The duration of the anorexia for PEG‐CCK9 was dose‐dependent, suggesting that PEGylation of CCK9 increases its circulation time. Both devazepide and 2‐NAP completely abolished the anorectic effect of i.p. PEG‐CCK9 indicating that its anorectic effect was solely due to stimulation of peripheral CCK1 receptors. British Journal of Pharmacology (2007) 152, 396–403; doi:10.1038/sj.bjp.0707390; published online 9 July 2007

Background and purpose: Cholecystokinin (CCK) stimulates the release of amylase and lipase from the normal pancreas. However, it is not clear to what extent this occurs in the early stages of pancreatitis induced by biliary tract obstruction in the rat and whether CCK initiates an inflammatory cascade in this condition. Experimental approach: Selective CCK1 receptor antagonists, JNJ‐17156516 ((S)‐(3‐[5‐(3,4‐dichloro‐phenyl)‐1‐(4‐methoxy‐phenyl)‐1H‐pyrazol‐3‐yl]‐2‐m‐tolyl‐propionic acid) and dexloxiglumide, were used to assess the response of plasma amylase and lipase to a CCK analogue, CCK8S, in normal rats and in rats with bile duct ligation. Key results: Both antagonists suppressed CCK8S‐induced elevation of plasma amylase activity in normal rats. JNJ‐17156516 was more potent than dexloxiglumide (ED50=8.2 vs >30 μmol kg−1 p.o.) and produced a longer lived inhibition (6 vs 2 h). Plasma amylase and lipase activity were elevated in parallel to CCK plasma concentrations after bile duct ligation and both activities were suppressed in a dose‐dependent manner by JNJ‐17156516 and dexloxiglumide. JNJ‐17156516 was ∼5‐ to 10‐fold more potent than dexloxiglumide. Infusion of CCK8S to naïve rats to achieve levels similar to those observed after bile duct ligation (20 pM) increased plasma amylase activity and activated nuclear factor‐κB in the pancreas. These effects were prevented by pretreatment with JNJ‐17156516. Conclusions and implications: The elevation of plasma amylase and lipase activity in the early stages of obstruction‐induced pancreatitis is largely driven by elevation of plasma CCK concentration and activation of CCK1 receptors. These data show that CCK is an initiating factor in acute pancreatitis in the rat. British Journal of Pharmacology (2008) 153, 1650–1658; doi:10.1038/bjp.2008.44; published online 25 February 2008

1 The full‐length, canine cholecystokinin 1 (CCK1) receptor was cloned from gallbladder tissue using RT–PCR with a combination of primers designed to interact with conserved regions of the human and rat CCK1 receptor, which also shared homology with the canine genomic sequence. 2 Analysis of the sequence of the canine CCK1 receptor revealed a 1287 base pair product, which encoded a 429 amino‐acid protein. This protein was 89% identical to the human and 85% identical to the rat CCK1 receptor. 3 The canine CCK1 receptor was expressed in CHO‐K cells for pharmacological characterization. In competition studies, using [125I]BH‐CCK‐8S as radioligand, the affinity values estimated for CCK receptor‐selective compounds were not significantly different between the canine and human CCK1 receptors (pKI±s.e.m. at canine CCK1 receptor; L‐364,718=8.82±0.08, L‐365,260=6.61±0.05, YF476=7.91±0.15, YM022=8.28±0.06 and dexloxiglumide=7.53±0.11). Furthermore, the selectivity of these compounds between canine CCK1 and CCK2 receptors was consistent with the selectivity between the human CCK1 and CCK2 receptors. 4 Two additional forms of the canine CCK1 receptor were identified during the cloning procedure. These had three (variant #1) and six (variant #2) amino‐acid differences from the wild‐type canine CCK1 receptor. Variant #1 bound [125I]BH‐CCK‐8S and displayed an identical pharmacological profile to the wild‐type receptor using the ligands described above. No significant binding was measured with variant #2. 5 In conclusion, we have cloned and pharmacologically characterized the canine CCK1 receptor. The data obtained will facilitate the interpretation of numerous pharmacological experiments that have been performed using canine tissue to elucidate the actions of CCK and gastrin. British Journal of Pharmacology (2005) 145, 374–384. doi:10.1038/sj.bjp.0706196

The pharmacology of the cholecystokinin CCK1 receptors endogenously expressed in human gallbladder and human ascending colon smooth muscle tissue was compared using radioligand binding assays. Saturation analysis of the interaction between the radiolabelled, selective CCK1‐receptor antagonist, [3H]‐L‐364,718, and enriched gastrointestinal tissue membranes suggested the presence of multiple binding sites in human colon but not human gallbladder. Competition studies, using a range of structurally diverse, CCK‐receptor selective ligands provided further evidence for CCK1 receptor heterogeneity in human colon tissue (nH values significantly less than unity for SR27897=0.77±0.07, 2‐NAP=0.73±0.03, YM220=0.70±0.09 and PD‐134,308=0.83±0.01). Moreover, the competition data for SR27897, 2‐NAP and YM220 were consistent with the interaction of these compounds at two binding sites. In contrast, in the human gallbladder assay, a single binding site model provided a good fit of the competition curve data obtained with all the CCK receptor selective compounds. The data obtained are consistent with the presence of a single CCK1 receptor binding site in the gallbladder but not in the colon. A two‐site analysis of the colon data, indicated that one of the two sites was indistinguishable from that characterized in the gallbladder. The molecular basis of the apparent receptor heterogeneity in the colon remains to be established. British Journal of Pharmacology (2002) 136, 873–882. doi:10.1038/sj.bjp.0704794