Gene symbol: LOC397740 (Caerulein Xenopus Laevis)

1. General Information

Caerulein is a` decapeptide with biological activity on GI smooth muscle contraction and pancreatic and gastric secretion.  It was identified based on its pharmacological action to lower blood pressure in extracts of the skin of the Australian Green Tree Frog (Figure 1) then known as Hyla caerulea (4) (Note 1). Most of the early research was centered in Italy and carried out by the research group of V. Erspamer. Subsequently caerulein was identified in other Australian Hylids, in the South American frog, Leptodactylus, and in the South African toad, Xenopus laevis (3, 10).  Sequencing of the peptide showed striking resemblance in amino acid sequence to the carboxyl terminus of cholecystokinin (CCK) and gastrin.  The amino acid sequence of caerulein and a related peptide, phyllocaerulein, from South American Hylid frogs (2) is shown in Figure 2.  Caerulein contains a sulfated tyrosine and the C-terminal seven amino acids are identical to CCK octapeptide except for a Thr residue substituting for Met.  Both peptides bear a carboxyl terminal amide group but caerulein also has a blocked amino terminal with pyroglutamine as the initial amino acid.  This blocking group reduces susceptibility to inactivation by amino peptidases.

Figure 1. Hyla Caerulea also known as the Australian Green Tree Frog. Image from en.wikipedia.org.

Figure 2. Amino acid comparison of Caerulein, Phyllocaerulein and CCK-OP. Pyr-Glu is pyroglutamate in which the free amino group of glutamic acid cyclizes to form a lactam.

Subsequent functional studies with purified and synthetic caerulein showed that its biological effects closely resembled that of CCK.  Both peptides were powerful stimulants of pancreatic secretion and gallbladder contraction (13).  Similar to CCK, these actions required the sulfation of the tyrosine residue (18).  In these early in vivo studies, caerulein was about three times as active as CCK purified from pig intestine (8).  The early availability of synthetic caerulein, named Caeruletide, for X-ray exam of the gallbladder led to its use in the 1970’s for pancreatic function studies in humans (14, 33).  Interestingly, cardiovascular effects were minimal at approved dosages suggesting that the prominent effect of skin extracts may have been due to another chemical in skin or usage of a supramaximal dose.

Caerulein precursors were found to be multiply represented in a Xenopus skin cDNA library (15, 26).  It was concluded that there was a family of precursors from several genes as well as alternative splicing (26).  For mature caerulein to be released, multiple processing steps must be involved similar to those involved in the synthesis of GI hormones.  In addition to cleaving the precursor into smaller fragments, specific enzymes must process the amino and carboxyl terminals.  Ultimately the peptides end up in secretory granules and are released into skin secretions.  The physiological role of caerulein and other frog skin peptides is unclear.  Most commonly it is speculated to repel predators or to act as an antibacterial agent.  How caerulein might have such action remains unclear.

2. Information on Caerulein and the Pancreas

As indicated above, caerulein appears to have a similar potency in vivo to natural porcine CCK-33 and synthetic CCK-8.  Similar to CCK, it primarily stimulates enzyme secretion and synergizes with secretin on fluid secretion in dog (23, 30), human (5, 9, 27), pigs (6), sheep (7).  In the rat, similar to CCK, caerulein stimulates both a low bicarbonate fluid secretion and robust digestive enzyme secretion (12).  In studies of the perfused rat pancreas, caerulein potentiated glucose stimulated insulin secretion and amylase secretion to levels similar to that of CCK-8 (28).   In vitro studies have shown that caerulein directly stimulates amylase release from isolated acinar cells and acini of guinea pig, rat and mouse pancreas (20, 29, 31, 35).  When compared, the magnitude of secretion was similar although caerulein was about twice as potent (17, 29).  Caerulein increased calcium mobilization and did not affect cAMP levels (11,20). A comparision of the ability of caerulein and CCK-8 to stimulate amylase release from isolated mouse pancreatic acini is shown in Figure 3. Note the similar bisphasic effect on secretion.

Caerulein appears to exert all of its biological effects on the pancreas through the CCK receptor.  Caerulein displaced ¹²⁵I-CCK binding from both acini and pancreatic membranes similar to CCK-8 although in some studies caerulein was more potent and in others less potent than CCK-8 (16, 29).  In a study of bullfrog brain and pancreas membranes, caerulein was 2-3 fold less potent than CCK-8 (32).  Because errors can result from contained water or other impurities affecting the potency, it is probably best to conclude that caerulein is comparable to CCK-8 in interacting with the CCK receptor and stimulating amylase secretion.

Figure 3. Comparison of dose-response curve for caerulein and CCK-8 to release amylase from isolated mouse pancreatic acini. Caerulein was obtained from Sigma and CCK-8 from Research Plus. Both were prepared as 10-4 M stock solutions in PBS plus 0.1 mg/ml bovine serum albumin accepting the suppliers’ statement of the mass of peptide supplied. Frozen aliquots were thawed and diluted into incubation medium.

The major use of caerulein in pancreatic studies over the last 25 years has been to induce a model of experimental pancreatitis.  In 1977 Horst Kern and colleagues showed that a continuous iv infusion of supramaximal caerulein into rats (5 μg/kg per hour) induced edematous pancreatitis with a tenfold increase in plasma amylase, and the appearance of large cytoplasmic vacuoles in pancreatic acinar cells (1, 19).  A more complete electron microscopic analysis was carried out by Watanabe et al (34).  In 1985, Niederau et al showed that repeated hourly ip injections of caerulein would induce a necrotizing pancreatitis in mice (24).  Subsequently several thousand papers have appeared using the caerulein model of acute pancreatitis primarily in rodents where most commonly 50 μg/kg is given in repeated doses.  Several reports have also appeared using dogs.  Studies in mice have been popular because of the availability of genetic models in this species.  Details of the method to induce acute pancreatitis have been presented elsewhere in the Pancreapedia (21).  Subsequently, repeated bouts of acute pancreatitis induced by caerulein have also been used to induce chronic pancreatitis (25) as well as a severe form of the disease (36).  One can ask why has caerulein been used so exclusively to induce pancreatitis rather than CCK?  It may simply have been that caerulein was more available in Europe at the time of the first studies and then other investigators simply followed suite.  By contrast, both CCK-8 and caerulein have been used in in vitro studies to induce acinar cell damage.  However, caerulein probably has an advantage in vivo in that its blocked amino terminal and replacement of Met residues likely results in a longer half life in the body which could be important especially with ip injections.  One piece of data in support of this was the observation by Mossner and colleagues that infusion of caerulein in humans compared to a derivative of CCK-9 without methione showed a threefold higher plasma level for comparable doses of caerulein when measured with a bioassay (22).  These authors concluded that caerulein was more stable against biological degradation than CCK.

3. Tools to study caerulein

a. Synthetic Peptide

Caerulein can be obtained from multiple sources including Sigma and Research Plus which we have used.

b. Antibodies

Because of similarity to the carboxyl terminal of CCK it would be difficult to measure caerulein by RIA although an antibody directed at the c terminal sulfated tyrosine might react with caerulein as well as CCK.  It can be measured by bioassay but the measurement would be the sum of CCK and caerulein.  An antibody to the carboxy terminal of CCK should localize caerulein in the frog skin.

4. Notes

1. This frog is now placed in the genus Litoria and is known as Litoria caerulea; at one time it was also called Rana caerulea.

5. References

1. Adler G, Hupp T, Kern H.F. Course and spontaneous regression of acute pancreatitis in the rat. Virchows Arch A Pathol Anat Histol 382: 31-47, 1979. PMID: 157597
2. Anastasi A, Bertaccini G, Cei J.M, De Caro G, Erspamer V, Impicciatore M. Structure and pharmacological actions of phyllocaerulein, a caerulein-like nonapeptide: its occurrence in extracts of the skin of Phyllomedusa sauvagei and related Phyllomedusa species. Br J Pharmac 37: 198-206, 1969. PMID: 5824931   
3. Anastasi A, Bertaccini G, Cei J.M, De Carro G, Erspamer V, Impicciatore M, Roseghini M. Presence of caerulein in extracts of the skin of Leptodactylus pentadactylus labyrinthicus and of Xenopus laevis. Br J Pharmacol 38: 221-229, 1970. PMID: 5413288
4. Anastasi A, Erspamer V, Endean R. Isolation and Amino Acid Sequence of Caerulein, the Active Decapepetide of the Skin of Hyla caerulea. Archives of Biochem and Biophys 125: 57-68, 1968. PMID: 5649531
5. Beglinger C, Kohler E, Stalder G.A, Jansen J, Gyr K. What is the maximal effective dose of caerulein in stimulating pancreatic secretion in man? Digestion 35: 125-128, 1986. PMID: 3770320.
6. Belloli C, Ormas P, Cissokho S, Beretta C. The effects of caerulein on exocrine pancreatic secretion in pigs. Vet Res Commun 6: 43-49, 1983. PMID: 6868347
7. Beretta C, Ormas P, Belloli C, Invernizzi A, Faustini R. Effects of Caerulein on exocrine pancreatic secretion in sheep. J Vet Pharmacol Ther 4: 219-224, 1981. PMID: 7349337
8. Bertaccini G, De Caro G, Endean R, Erspamer V, Impicciatore M. The action of caerulein on pancreatic secretion of the dog and Biliary secretion of the dog and the rat. Br J Pharmac 37: 185-197, 1969. PMID: 5824930
9. Cavallini G, Mirachian R, Angelini G, Vantini I, Vaona B, Bovo P, Gelpi F, Ederle A, Dobrilla G, Scuro L.A. The role of caerulein in tests of exocrine pancreatic function. Scand J Gastroenterol 13: 3-15, 1978. PMID: 345411
10. De Caro G, Endean R, Erspamer V, Roseghini M. Occurrence of Caerulein in Extracts of the Skin of Hyla Caerulea and Other Australian Hylids. Br. J. Pharmac. Chemother 33: 48-58, 1978. PMID: 5660165
11. Deschodt-Lanckman M, Robberecht P, De Neff P, Lammens M, Christophe J. In Vitro Action of Bombesin and Bombesin-Like Peptides on Amylase Secretion, Calcium Efflux, and Adenylate Cyclase Activity in the Rat Pancreas.  J Clin Invest 58: 891-898, 1976. PMID: 184111
12. Dockray G.J. The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. J Physiol 225: 679-692, 1972. PMID: 5076393
13. Erspamer V. Progress Report: Caerulein. Gut 11: 79-87, 1970. PMID: 4907841
14. Gullo L, Costa P.L, Fontana G, Labo G. Investigation of exocrine pancreatic function by continuous infusion of caerulein and secretion in normal subjects and in chronic pancreatitis. Digestion 14: 97-107, 1976. PMID: 950084
15. Hoffmann W, Bach T.C, Seliger H, Kreil G. Biosynthesis of caerulein in the skin of Xenopus laevis: partial sequences of precursors as deduced from cDNA clones. EMBO J 2: 111-114, 1983. PMID: 11894896
16. Innis R.B, Snyder S.H. Distinct cholecystokinin receptors in brain and pancreas. Proc Natl Acad Sci USA 77: 6917-6921, 1980. PMID: 6256771
17. Jensen R.T, Lemp G.F, Gardner J.D. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Natl Acad Sci USA 77: 2079-2086, 1980. PMID: 6246521
18. Johnson L.R, Stening G.F, Grossman M.I. Effect of Sulfation on the Gastrointestinal Actions of Caerulein. Gastroenterology 58: 208-216, 1970. PMID: 4904949
19. Lampel M, Kern H.F. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue.  Virchows Arch A Pathol Anat Histol 373: 97-117, 1977. PMID: 139754
20. May R.J, Conlon T.P, Erspamer V, Gardner J.D. Actions of peptides isolated from amphibian skin on pancreatic acinar cells. Am J Physiol 235: E112-E118, 1978. PMID: 210673
21. Mayerle J, Sendler M, Lerch M.M. Secretagouge (Caerulein) induced pancreatitis in rodents. The Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2013.2.
22. Mossner J, Sprenger C, Secknus R, Kestel W, Fischbach W. Comparison between the synthetic cholecystokinin analogues caerulein and Thr²⁸NLE³¹CCK_25-33(CCK₉) with regards to plasma bioactivity, degradation rate and stimulation of pancreatic exocrine function. Z Gasterotenterol 29: 59-64, 1990. PMID: 1714671
23. Nakajima S. The action of the C-terminal octapeptide of cholecystokinin and related peptides on pancreatic exocrine secretion. Gut 14: 607-615, 1973. PMID: 4743491
24. Niederau C, Ferrell L.D, Grendell J.H. Caerulein-induced acute necrotizing pancreatitis in mice: protective effects of proglumide, benzotript, and secretin. Gastroenterology 88: 1192-1204, 1985. PMID: 2984080
25. Reed A, Gorelick F. Animal Models of Chronic Pancreatitis. The Pancreapedia: Exocrine Pancreas Knowledge Base, DOI: 10.3998/panc.2014.1.
26. Richter K, Egger R, Kreil G. Sequence of Preprocaerulein cDNAs Cloned from Skin of Xenopus laevis. J Biol Chem 8: 3676-3680, 1986. PMID: 3753978 
27. Robberecht P, Cremer M, Vandermeers A, Vandermeers-Piret M.C, Cotton P, De Neef P, Christophe J. Pancreatic secretion of total protein and of three hydrolases collected in healthy subjects via duodenoscopic cannulation. Effects of secretin, pancreozymin, and caerulein. Gastroenterology 69: 374-379, 1975. PMID: 1150045
28. Sakamoto C, Otsuki M, Ohki A, Yuu H, Maeda M, Yamasaki T, Baba S. Glucose-dependent insulinotropic action of cholecystokinin and caerulein in the isolated perfused rat pancreas. Endocrinol  110: 398-402, 1982. PMID: 6173205
29. Sankaran H, Goldfine I.D, Deveney C.W, Wong K.Y, Williams J.A. Binding of Cholecystokinin to High Affinity Receptors on Isolated Rat Pancreatic Acini. J of Biol Chem 255: 1849-1853, 1980. PMID: 6243650
30. Tardini A, Anversa P, Bordi C, Bertaccini G, Impicciatore M. Ultrasctructural and Biochemical Changes after Marked Caerulein Stimulation of the Exocrine Pancreas in the Dog. Am J Path 62: 35-48, 1971. PMID: 5538718
31. Uhlemann E.R, Rottman A.J, Gardner J.D. Actions of peptides isolated from amphibian skin on amylase release from dispersed pancreatic acini. Am J Physiol 236: E571-E576, 1979. PMID: 443378
32. Vigna S.R, Steigerwalt R.W, Williams J.A. Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas. Reg Peptides 9: 199-212, 1984. PMID: 6098940
33. Vincent M.E, Wetzner S.M, Robbins A.H. Pharmacology, Clinical Uses, and Adverse Effects of Ceruletide, A Cholecystokinetic Agent.  Pharmacotherapy 2: 223-234, 1982. PMID: 6763205
34. Watanabe O, Baccino F.M, Steer M.L, Meldolesi J. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis. Am J Physiol 246: G457-G467, 1984. PMID: 6720895
35. Williams J.A, Korc M, Dormer R.L. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. Am J Physiol Endocrinol 4: E517-E524, 1978. PMID: 215042
36. Zhang H, Neuhofer P, Song L, Rabe B, Lesina M, Kurkowski MU, Treiber M, Wartmann T, Regner S, Thorlacius H, Saur D, Weirich G, Yosimura A, Halangk W, Mizgerd JP, Schmid RM, Rose-John S, Algül H.    IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality. J Clin Invest 123:1019-1031, 2013. PMID: 23426178