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.

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.

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.