MOLECULE PAGE Cholecystokinin Rashmi Chandra and Rodger A. Liddle Department of Medicine. Duke University Medical Center and Department of Veterans Affairs Durham, NC 27710 e-mail: [email protected] Version 1.0, July 27, 2018 DOI: Gene Symbol: CCK Abstract In 1928, Ivy and Oldberg discovered that intestinal lumen. The basolateral membrane often contains extracts prepared after instilling weak acid or fats one or more basal process(es) named neuropods into the proximal duodenum, elicited gallbladder that run alongside or project into the lamina propria contraction in dogs, cats, and guinea pigs (33). (6, 10). Neuropods contain neuronal markers and Based on this biological property, the hormone was have been shown to interact with enteric nerves named cholecystokinin (CCK). In addition to suggesting that in addition to secretion to the gallbladder contraction, CCK was later shown to blood, CCK can be released directly adjacent to stimulate pancreatic secretion (55) and to delay enteric nerves (Figure 3). CCK immunoreactivity is gastric emptying by its effect on the lower abundant in the pyloric region of mouse stomach esophageal sphincter (80). CCK was the first (45), cerebral cortex, dopaminergic neurons hormone shown to influence satiety and cause projecting to the limbic forebrain and ventromedial reduction in food intake (23). Due to this discovery hypothalamus, peripheral nerves of the and the implications of CCK’s therapeutic potential gastrointestinal tract, celiac plexus, and vagus for eating disorders, considerable attention has nerve (2, 48). CCK has been shown to function focused on the study of this hormone. both as a hormone and a neurotransmitter and belongs to the ‘brain-gut’ family of peptides. In In the gastrointestinal tract, CCK is secreted by addition to intestinal and neuronal expression, discrete enteroendocrine cells (EECs) which CCK is also expressed in other tissues such as the contain intermediate-size secretory granules (I urogenital tract and heart (78). The structure of cells) (95). CCK-producing cells are primarily CCK and its function, pertaining to its role in the located in the proximal small intestine (duodenum gastrointestinal tract, is discussed in this review. and jejunum, Figure 1), and their numbers decrease significantly towards the distal end (ileum and colon) (Figure 2). CCK cells are often flask- shaped with the narrow apical edge facing the gut This work is subject to a Creative Commons Attribution 3.0 license. Figure 3. Transverse section of CCK-EGFP mouse duodenum showing EGFP positive CCK cells (green) and enteric nerves immunostained for pan-neuronal marker PGP9.5 (red). Two CCK cells from the left panel, Cells A and B, are shown at higher magnification on the right. Cell A has three short whisker-like neuropods and its basolateral surface is in contact with enteric nerves. Figure 1. Transverse section of mouse duodenum Cell B has 2 thin neuropods (arrows). The longer of the showing CCK cells (green). Nuclei are stained with two neuropods terminates in a bulb and is in contact with DAPI (blue). a nerve. 1. General CCK is present in all vertebrates from fish to mammals. A CCK-like peptide has been found in the protochordate Ciona intestinalis, suggesting that the CCK/gastrin family probably arose 500 million years ago (37). Based on the phylogeny of CCK and gastrin genes in protochordates versus cartilaginous fish such as Squalus acanthias, and amphibians, it is proposed that gene duplication occurred 350 million years ago during the appearance of cartilaginous fish (37, 38). In humans, the CCK gene is present on chromosome 3, spans 7 kb, and consists of three exons, the first of which is noncoding (87, 88). The mouse Cck gene is similar in structure to the human gene and Figure 2. The number of CCK cells is highest in the is present on chromosome 9 in a syntenic cluster proximal small intestine of the mouse and decreases (21). exponentially towards the distal end (ileum). CCK antibody (8) used for immunostaining sections did not react with gastrin. Number of cells in 5 sections spread CCK polypeptides of various lengths have been over 1 inch in length were counted (R. Chandra, described in the literature (Figure 4). Although unpublished data). there are four known transcripts of the human CCK gene, only a single preprocholecystokinin 2 polypeptide of 115 amino acids is synthesized. gastrin which is present at much higher After proteolytic excision of the signal peptide by concentrations in the blood (5). signal peptidase, procholecystokinin of 94 amino acids is generated. This is again cleaved on both Regulation of CCK secretion the N (24 amino acids) and C (12 amino acids) termini by endopeptidase and proprotein CCK is released from EECs in response to entry of convertase 1 respectively, to generate a mid- food into the duodenum. Plasma levels of CCK section polypeptide of 58 amino acids known as increase from basal levels of 0.5-1 pM to peak CCK-58, which is the largest known circulating levels of 5-15 pM within a few minutes of food form of the hormone (19). It contains a carboxyl- ingestion. In rodents, peak plasm levels are usually amidated phenylalanine and O-sulfated tyrosine attained within 20 minutes of oral gavage. In residue, which is responsible for increasing its humans, postprandial levels remain elevated for 3- biological activity by approximately 100-fold (18, 5 hours until food empties from the stomach into 76, 77). CCK-58 undergoes subsequent the duodenum (57). Therefore, gastric emptying endopeptidase cleavage at single or double basic affects CCK secretion. Plasma CCK levels decline residues to generate shorter peptides, CCK-39, once food passes from the proximal small intestine. CCK- 33, CCK-22, CCK-12 and CCK-8 (3, 84). The half-life of CCK in the plasma is very short; in CCK-8 is the smallest peptide which exhibits dogs the half-life of CCK-58 was 4.4 ± 0.6 minutes complete biological activity and is used most often and that of CCK-8 was shown to be 1.3 ± 0.1 in experiments for assessing CCK function. The minutes (32). CCK is cleared from the circulation five C-terminal residues of CCK are identical to as it passes through the liver and by neutral gastrin, and as a result these two hormones display endopeptidases in capillary endothelial cells (71). some functional similarities. This sequence identity CCK secretion is stimulated by ingested fats, complicated the measurement of CCK in the blood, proteins, and amino acids, whereas carbohydrates as many antibodies against CCK-8 cross react with such as glucose cause only a brief, transient increase in circulating CCK levels (57). Figure 4. Amino acid structure of the human CCK precursor and the different forms of CCK produced by processing. 3 The apical surface of CCK-producing cells is appears to be mediated by an endogenous exposed to the intestinal lumen and receptors protease-sensitive CCK-releasing peptide in the located on the apical surface can be stimulated by intestinal lumen (30, 50, 85). In addition to food molecules present in the lumen. Aromatic L- proteases, bile acids in the intestine affect CCK amino acids such as phenylalanine and tryptophan secretion (26, 55). In rats, luminal administration of (but not non-aromatic amino acids such as alanine) taurocholate inhibited pancreatic enzyme secretion stimulate CCK release through a Ca2+-dependent as well as CCK (90). In humans, single bile acids mechanism mediated by the calcium-sensing did not cause a decrease in CCK release, however, receptor (CaSR) (61, 94) while L-phenylalanine, L- under conditions in which endogenous release of leucine, and L-glutamic acid mediate CCK release bile acid was inhibited by the CCK1 receptor through umami taste receptors T1R1 – T1R3 (14). antagonist loxiglumide, addition of a mixture of bile In addition to amino acids, medium to long chain acids to a test meal prevented the increase in CCK fatty acids (C12 and longer) also stimulate CCK release suggesting that bile acids play an important release (65). The long chain fatty acid receptor role in downregulating CCK secretion (43). GPR40 also known as free fatty acid receptor 1 (FFAR1), mediates some fatty acid-dependent CCK released from EECs can act locally via a CCK secretion (62). Fat meditated CCK- paracrine mechanism or enter the enteric blood stimulation was completely eliminated in stream and exert effects on distant target organs immunoglobulin-like domain containing receptor 1 through hormonal mechanisms. There is evidence (ILDR1) knockout mice suggesting that ILDR1 for neural activation of vagal afferents in the plays a role in CCK release (13). ILDR1-mediated intestinal mucosa which express CCK1 receptors CCK release occurred only in the presence of both and terminate in the lamina propria (74). Although high density lipoprotein (HDL) and fatty acids, the effect of CCK on the vagus nerve was believed suggesting a novel pathway in which uptake of to be paracrine or hormonal action, recently, CCK HDL and/or fatty acid from the basolateral cells have been shown to be in direct contact with membrane could play an important role in CCK neurons (7, 12, 53) and this connection may release. provide a direct neural link between the gut and brain. Evidence is accumulating that cell surface receptors linked to hormone secretion may be Cholecystokinin Receptors located on the basolateral surface of the CCK cell. ILDR1-mediated CCK release requires both fatty The action of CCK on tissues is mediated by two G acids and HDL which are most likely secreted onto protein-coupled receptors, CCK1 and CCK2, the basolateral surface of the intestinal epithelium formerly known as CCK-A (for alimentary) and suggesting that CCK cells respond to absorbed CCK-B (for brain) (17). CCK1 receptors are mainly nutrients (13).