Cellular and subcellular localization of cholecystokinin (CCK)-1 receptors in the pancreas, gallbladder, and stomach of mice.

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.

Cholecystokinin receptor antagonist halts progression of pancreatic cancer precursor lesions and fibrosis in mice.

OBJECTIVES: Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. METHODS: The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. RESULTS: Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001). CONCLUSIONS: These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

Expression profile of cholecystokinin type-A receptor in gallbladder cancer and gallstone disease.

BACKGROUND: Regulatory peptide receptors have attracted the interest of oncologists as a new promising approach for cancer pathology, imaging and therapy. Although cholecystokinin (CCK) is a potent modulator of gallbladder contractility and plays a potential role in pancreatic carcinogenesis through CCK type-A receptor (CCKAR), its role in gallbladder cancer (GBC) is still unknown and immunohistochemical detection of CCKAR in the gallbladder has not yet been reported. This novel case-control study aimed to investigate the expression profile of CCKAR in GBC and gallstone disease (GSD). METHODS: This study included 162 samples of gallbladder: 94 from GBC and 68 from GSD. Expression of CCKAR was analyzed by immunohistochemistry and immunoblotting. The results were statistically correlated with disease history including age, sex, presence of gallstone, stage and differentiation. RESULTS: CCKAR was positive in 30/68 (44.1%) of GSD and 72/94 (76.6%) of GBC samples. Fifty-one of the 72 (70.8%) CCKAR-positive GBC samples showed over-expression. Interestingly, consistent results also appeared in the immunoblotting study. CONCLUSIONS: CCKAR expression was significantly increased in GBC compared to GSD. Moreover, CCKAR expression was associated with the degree of tumor differentiation, i.e., less expression in poorly-differentiated tumors. Thus, it has future prognostic and therapeutic implications in the management of GBC.

Immunohistochemical localization of CCK1 cholecystokinin receptors in normal and neoplastic human tissues.

BACKGROUND: The biological effects of cholecystokinin (CCK) are mediated by two distinct G protein-coupled receptors, CCK1 and CCK2. Although it is well established that CCK receptors are widely distributed throughout the normal gastrointestinal tract, little is known about their cellular and subcellular localization in human normal and neoplastic tissues. METHODS: We developed and characterized a novel antipeptide antibody to the carboxyl-terminal region of the human CCK1 receptor. Specificity of the antiserum was demonstrated by 1) detection of a broad band migrating at a relative molecular mass of 85,000-95,000 in Western blots of membranes from CCK1-expressing tumors and CCK1-transfected cells, 2) cell surface staining of CCK1-transfected cells, 3) translocation of CCK1 receptor immunostaining after agonist exposure, and 4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. The distribution of CCK1 receptors was investigated in 74 human tumors and their tissues of origin. RESULTS: The presence of CCK1 receptors was rarely detected in human tumors except for carcinoids, insulinomas, pituitary adenomas, and meningiomas. CCK1 receptors were clearly located at the plasma membrane and uniformly present on nearly all tumor cells. In the gastrointestinal tract, CCK1 receptor immunoreactivity was highly abundant in chief cells of the gastric mucosa, in myenteric ganglion cells, and in myenteric nerve fibers. CONCLUSION: This is the first localization of CCK1 receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The overexpression of CCK1 receptors in a subset of human neuroendocrine tumors may provide a molecular basis for efficient targeting of these tumors with radiolabeled CCK analogs.

Bile-pancreatic juice exclusion increases cholinergic M3 and CCK-A receptor expression and interleukin-6 production in ligation-induced acute pancreatitis.

BACKGROUND: Using an original model, the Donor Rat Model, we showed that bile-pancreatic juice (BPJ) exclusion from gut exacerbates ligation-induced acute pancreatitis in rats. We also showed that muscarinic cholinergic M3 and CCK-A receptor expression is induced following duct ligation. Increased receptor number potentially could exacerbate cytokine production. We hypothesize that BPJ exclusion is responsible for M3 and CCK-A receptor induction and increased interleukin-6 (IL-6) production. METHODS: M3 and CCK-A receptor expression and IL-6 production were compared in rat pancreata 1 to 3 hours after duct ligation with or without BPJ replacement. RESULTS: Our studies showed that BPJ replacement attenuates duct ligation-induced increases in M3 and CCK-A receptor expression and IL-6 production. CONCLUSIONS: In this model, BPJ exclusion from gut induces M3 and CCK-A receptor expression and increases IL-6 production. In this experimental corollary of gallstone pancreatitis, BPJ exclusion from gut may play a key role in the mechanism of disease pathogenesis.

Immunolocalization of CCK1R in rat brain using a new anti-peptide antibody.

An antibody directed at the carboxy tail of the cholecystokinin-1 receptor (CCK1R) was characterized by ELISA and Western blotting. Immunohistochemistry established that CCK1R-like immunoreactivity (CCK1R-LI) was widely and topographically distributed through the neuroaxis, appearing relatively higher in rhi- and diencephalon, and intense in both neuronal somata (cytoplasmic) and processes. CCK1R-LI was found in new loci, but also in areas previously identified by receptor autoradiography, electrophysiology and in situ hybridization of CCK1R mRNA. The widespread distribution of CCK1R has implications for the functional roles of these receptors in brain. The high titre and low background seen with this new antiserum makes it of great value for cell and tissue research.