Cholecystokinin (CCK) is member of a family of regulatory peptides with a remarkably well preserved C-terminal sequence (1–3). The family also includes frog skin peptides (caerulein and phyllocaerulein) and the protochordean neuropeptide cionin, but in mammals, CCK and gastrin are the only family members (Figure 1).

After the discovery in 1928 (6), CCK became part of the classical troika of gut hormones together with secretin and gastrin. The last decades, however, have shown that CCK, in addition to its local acute functions in digestion (gallbladder emptying and pancreatic enzyme secretion), is also a growth factor, a neurotransmitter in the brain and peripheral neurons [for reviews, see Ref. (7–9)], and besides, it may be a spermatozoan fertility factor, a natriuretic kidney peptide, an anti-inflammatory cytokine in the immune system, and a cardiac marker of heart failure. The long history has made the CCK literature comprehensive and at some points also confusing because impure CCK preparations with little attention paid to species differences and to physiological levels were used initially. In addition, most assays for measurement of CCK in plasma and elsewhere lacked specificity and sensitivity (10–12).

The biochemical concept of CCK as a single hormonal peptide from the small intestine has also changed considerably. Now CCK is known to be synthetized and released in multiple molecular forms. And the CCK gene is expressed at peptide level in a cell-specific manner in neurons, endocrine cells, and epithelial cells outside the gastrointestinal tract (Table 1). All known biological effects of CCK peptides reside in the conserved C-terminal heptapeptide sequence (Figure 1). Modification of this sequence grossly reduces or abolishes receptor binding and biological effects (13–15). The N-terminal extensions of the common C-terminus increase the biological potency and the specificity for receptor binding. Of particular importance is the tyrosyl residue in position seven [as counted from the C-terminus (Figure 1)]. The tyrosyl residue is rarely completely sulfated (16–20). The CCK₂ receptor binds sulfated and unsulfated ligands equally well, whereas the CCK₁ receptor is exclusive and requires Y-sulfation of the ligand.

The following is a short review about the biology of CCK with emphasis on the recently recognized widespread expression (Table 1) and besides an update on the classic gastrointestinal effects of CCK peptides.

As described earlier (9), “the exomal unit of the CCK gene is seven kilobases interrupted by two introns (23). The first of the three exons is small and non-coding. Several conserved regulatory elements have been identified in first 100 bp of the promoter, including an E-box element, a combined cAMP response element (CRE)/12-O-tetradeconoylphorbol-13-acetate response element (TRE), and a GC-rich region (24, 25). Whereas the function of the E-box and the GC-rich region is not fully clarified (26, 27), the combined CRE/TRE sequence plays an important role in the regulation of CCK transcription. The CRE/TRE binds the transcription factor CREB, which is activated by phosphorylation by several signaling pathways, including cAMP, fibroblast growth factor, pituitary adenylate cyclase-activating polypeptide, calcium, hydrolyzates, and peptones to ultimately induce CCK transcription (28–32). Only one CCK mRNA molecule has been found, and the CCK peptides are thus fragments of the same proCCK protein. The mRNA has 750 bases, of which 345 are protein coding (33, 34). The concentrations of CCK mRNA in cerebrocortical tissue are similar to that of the duodenal mucosa (34), and in the brain, there is a rapid synthesis of CCK peptides (35).

The primary translational product, preproCCK, has 115 amino acid residues. The first part is the signal peptide. The second part with considerable species variation is a spacer peptide. The bioactive CCK peptides are derived from the subsequent 58 amino acid residues (16, 18, 36–38), and the species variation is small in this sequence. The processing of proCCK is cell-specific: endocrine cells contain a mixture of the medium-sized CCK-58, -33, -22, and -8, whereas neurons mainly release CCK-8 and to some extent CCK-5 (16, 39). The endoproteolysis of proCCK occurs mainly at monobasic sites. Y-77 is mostly O-sulfated (16–20, 40), which is decisive for CCK₁ receptor binding.

In the small intestine, CCK peptides are synthesized in endocrine I-cells (41), whose apical membrane is in contact with the intestinal lumen and whose basal region contains secretory granules with CCK peptides. CCK is also synthesized in pituitary corticotrophs and melanotrophs, in thyroid C-cells (17), and in adrenal medullary cells (42, 43). In the pituitary cells, CCK constitutes a small fraction of the hormones. Tumors originating from pituitary corticotrophs, however, produce larger amounts of CCK (44).”

It is the brain that expresses most CCK (16, 39, 42). Moreover, cerebral CCK neurons are more abundant than neurons of other neuropeptides (42, 45, 46). While most peptidergic neurons occur in subcortical regions, CCK is expressed in the highest concentrations in neocortical neurons (39, 42, 47). The perikarya of the cortical CCK nerves are distributed in layers II–VI, with the highest frequency in layers II and III (42, 48). CCK in mesencephalic dopamine neurons projecting to the limbic area of the forebrain (45) has aroused clinical interest because these neurons are supposed to be involved in schizophrenia.

Outside the brain, the colon contains numerous CCK neurons, whereas jejunum and ileum are less innervated (42). Colonic CCK fibers occur in the circular muscle layer, which they penetrate to form a plexus in the submucosa (42). In accordance with these locations, CCK peptides excite colonic smooth muscles and release acetylcholine from neurons in both plexus myentericus and submucosa (49). Ganglionic cell somas in pancreatic islets are also surrounded by CCK nerves (50). Moreover, CCK nerve terminals also surround pancreatic islets (51). Finally, afferent vagal nerve fibers also contain CCK (52, 53).

Also mentioned before (9), “CCK in circulation originates mainly from intestinal endocrine cells. The release to blood was not possible to examine until specific assays were developed (10–12, 54). The assays have confirmed that protein- and fat-rich food is the most important stimulus (11, 54). Of the constituents, protein and l-amino acids as well as digested fat cause significant CCK release (54, 55). Carbohydrates only release small amounts of CCK (54), but hydrochloric acid also stimulates release (55).

The release from neurons has been examined directly in brain slices and synaptosomes (56, 57). Potassium-induced depolarization caused a calcium-dependent release of CCK-8. Similarly, depolarization releases CCK peptides from the hypothalamic dopamine neurons that innervate the intermediate lobe of the pituitary (58).

By analogy with other neuropeptides, it is possible that overflow from peripheral CCK neurons may contribute slightly to CCK in plasma.By comparison with identified CCK peptides, it has been possible to deduce the molecular pattern of CCK in plasma. The picture has varied (12) due to species differences and because the molecular pattern along the gut varies (59, 60). Furthermore, the distribution may vary during stimulation. In man, CCK-33 predominates in plasma, but CCK-58, -22, and -8 are also present (11, 61).

In the basal state, the concentration of CCK in plasma is around 1 pmol/l, but often less. The concentration increases within 20 min to 3–5 pmol/l during meal stimulation, and then declines gradually only to reach a second peak after 1.5–2 hours. In comparison with most other pancreatic and gastrointestinal hormones (62), the concentrations of CCK in plasma are low. When food-induced CCK in plasma is mimicked by infusion of exogenous CCK, the same degree of gallbladder contraction and release of enzymes as seen during meals occurs (54, 62–64). Therefore, the low circulating concentrations of CCK are sufficient to account for the gallbladder contraction and pancreatic enzyme secretion during meals.

Because the cholecystokinetic and pancreozymic potency of CCK-33 and CCK-8 on a molar base are identical (65), it may seem less important what I-cells release during digestion.” On the other hand, CCK-58, -33, and -22 are cleared from blood at a significantly slower rate than CCK-8.

The cellular effects of CCK peptides are mediated via two receptors (66, 67). The “alimentary” CCK-A or CCK₁ receptor (66) mediates gallbladder contraction, relaxation of the sphincter of Oddi, pancreatic growth and enzyme secretion, delay of gastric emptying, and inhibition of gastric acid secretion via fundic somatostatin (68). CCK₁ receptors have been found also in the anterior pituitary, the myenteric plexus, and areas of the midbrain (69, 70). The CCK₁ receptor binds with high affinity CCK peptides that are amidated and sulfated, whereas the affinity for non-sulfated CCK peptides and gastrins is negligible.

The CCK-B or CCK₂ receptor (the “brain” receptor) is the predominant CCK receptor in the brain (67, 71). It is less specific than the CCK₁ receptor and binds also non-sulfated CCK, gastrins, and C-terminal fragments such as CCK-5. It has been shown that the gastrin receptor cloned from the stomach (67) and CCK₂ receptors are identical (71, 72). The gastrin/CCK₂ receptor is expressed also in substantial amounts in pancreatic islet cells in man (73).

The defining functions of CCKs in digestion have been detailed regularly [for instance, see Ref. (6, 7)].

The major sites of CCK expression are as mentioned endocrine cells in the gut, the brain, and in peripheral nerves. But the last decades have uncovered additional sites and cell types that also express the CCK gene at peptide level (Table 1). In some of these sites, proCCK is not processed to the known α-amidated peptides. Their functions are therefore still unknown. But since CCK receptors also have such widespread expression (66, 67, 70–73, 94, 95), there is both room and need for delineation of the roles of CCK released from the “new” sites.

Since the identification of CCK half a century ago as a single peptide with a sequence of 33 amino acid residues (CCK-33), the CCK story has been loaded with major revelations: first, it was shown that the C-terminus of CCK was similar to that of gastrin, and that CCK and gastrin peptides share the same receptor, the CCK₂ receptor. Then, it was demonstrated that bioactive CCK occurs in multiple molecular forms—from CCK-58 to CCK-5 with and without tyrosyl O-sulfations. At variable intervals, it has since been shown that CCK peptides are expressed all over the body: in central and peripheral neurons, in intestinal and extraintestinal endocrine cells, in germ cells, kidney epithelial cells, cardiac myocytes, and immune cells. Moreover, the proCCK maturation appears to be cell specific, and tumors expressing CCK release correspondingly varying multifaceted patterns of CCK peptides. Thus, today CCK should be seen as an almost ubiquitous system of intercellular messenger peptides. The complex biology is probably characteristic for many regulatory peptides, for which the CCK system may serve as a source of inspiration for further research.

The author confirms being the sole contributor of this work and approved it for publication.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.