Cholecystokinin (CCK) Assays

David G. Trist1, Mauro Corsi1

1 Glaxo Wellcome S.p.A. Medicines Research Centre, Verona, Italy
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.13
DOI:  10.1002/0471141755.ph0413s04
Online Posting Date:  May, 2001
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Abstract

Cholecystokinin (CCK) is a peptide that acts as a peripheral hormone and as a central neurotransmitter. To date, two distinct receptors have been identified for CCK using structural and operational criteria; CCK1 and CCK2 (formerly CCKA and CCKB, respectively). In addition, there is the gastrin receptor found in the stomach which has a high structural similarity to the CCK2 receptor, but which displays a different pharmacology. This unit presents two methods for the quantification of selective agonists and antagonists at CCK1 receptors and an assay for CCK2 receptors. In the first tissue, the guinea‐pig ileum longitudinal muscle with myenteric plexus, both CCK1 and CCK2 receptors are present in the same preparation. Each receptor is distinguished in this assay using selective agonists and antagonists against the unwanted receptor subtype. The second preparation, the guinea‐pig gallbladder, is a classical preparation for studying CCK1 receptor‐active compounds in the absence of CCK2 sites

     
 
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Table of Contents

  • Basic Protocol 1: Isolated Ileum Longitudinal Muscle/Myenteric Plexus (LMMP) Preparation
  • Basic Protocol 2: Gall Bladder Preparation
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolated Ileum Longitudinal Muscle/Myenteric Plexus (LMMP) Preparation

  Materials
  • Male guinea pigs (350 to 400 g)
  • Physiological salt solution (PSS; see recipe)
  • Carbogen gas (5% CO 2/95% O 2) in tank equipped with regulator capable of delivering gas under extremely low pressure (i.e., 0.8 to 1.0 bar)
  • 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP; Sigma; prepare fresh daily in distilled water as stock solution appropriate for adding to organ bath at 30 µm final concentration)
  • Test compounds (see recipe for representative compounds)
  • Petri dish equipped with a fine capillary tube through which Carbogen gas can be bubbled into the solution
  • Surgical instruments:
  •  Scissors
  •  Forceps
  •  Blade
  •  2.5‐ml syringe
  •  6‐mm diameter glass pipet
  •  Cotton‐tipped applicators
  • Apparatus for measuring tissue responses (Fig. ) including:
  •  5‐G fine silk thread
  •  Isotonic tissue holder, equipped with means for finely adjusting resting tension (Ugo Basile Biological Apparatus; also see unit 4.2)
  •  Thermostatted circulating water bath
  •  Heated isolated organ bath capable of being bubbled continuously with Carbogen gas and being rapidly emptied and filled with warmed PSS
  •  Chart recorder (Ugo Basile Biological Apparatus)
  • Additional reagents and equipment for measuring length‐tension relationships in isolated tissue (unit 4.2)

Basic Protocol 2: Gall Bladder Preparation

  Materials
  • Male guinea pigs (350 to 400 g)
  • Physiological salt solution (PSS; see recipe)
  • Carbogen gas (5% CO 2/95% O 2) in tank equipped with regulator capable of delivering gas under extremely low pressure (i.e., 0.8 to 1.0 bar)
  • 100 mM KCl
  • Test compounds (see recipe for representative compounds)
  • Petri dish equipped with a fine capillary tube through which Carbogen gas can be bubbled into the solution
  • Surgical instruments:
  •  Scissors
  •  Forceps
  •  Blade
  •  Cork board
  •  2.5‐ml syringe
  •  6‐mm diameter glass pipet
  •  Cotton‐tipped applicators
  • Apparatus for measuring tissue responses (Fig. ) including:
  •  5‐G fine silk thread
  •  Isometric tissue holder, equipped with means for finely adjusting resting tension (Ugo Basile Apparatus; also see unit 4.2)
  •  Thermostatted circulating water bath
  •  Heated isolated organ bath capable of being bubbled continuously with Carbogen gas and being rapidly emptied and filled with warmed PSS
  •  Chart recorder (Ugo Basile Apparatus)
  • Additional reagents and equipment for measuring length‐tension relationships in isolated tissue (unit 4.2)
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Figures

Videos

Literature Cited

Literature Cited
   Arunlakshana, O. and Schild, H.O. 1959. Some quantitative uses of drug antagonists. Br. J. Pharmacol. 14:48‐58.
   Corsi, M., Pojani, G., Dal Forno, G., Pietra, C., Gaviraghi, G., and Trist, D. 1993. Analysis of the CCKB receptor antagonism of virginiamycin in guinea‐pig ileum longitudinal myenteric plexus. Br. J. Pharmacol. 108:1164‐1168.
   Corsi, M., Palea, S., Pietra, C., Oliosi, B., Gaviraghi, G., Sugg, E., Van Amsterdam, F.T.M., and Trist, D.G. 1994. A further analysis of the contraction induced by activation of cholecystokinin A receptors in guinea pig isolated ileum longitudinal muscle‐myenteric plexus. J. Pharmacol. Exp. Ther. 270:734‐740.
   Costall, B., Domeney, A.M., Hughes, J., Kelly, M., Naylor, R.J., and Woodruff, G.N. 1991. Anxiolytic effects of CCKB antagonists. Neuropeptides 19:65‐73.
   Dal Forno, G., Pietra, C., Urciuoli, M., Van Amsterdam, F.T.M., Toson, G., Gaviraghi, G., and Trist, D. 1992. Evidence for two cholecystokinin receptors mediating the contraction of the guinea pig isolated ileum longitudinal muscle myenteric plexus. J. Pharmacol. Exp. Ther. 261:1056‐1063.
   De Lean, A., Munsen, P.J., and Rodbard, D. 1978. Simultaneous analysis of families of sigmoidal curves:Application to bioassay, radioligand assay and physiological dose‐response curves. Am. J. Physiol. 235:E97‐E102.
   Deschenes, R.J., Lorenz, L.J., Huan, R.S., Roos, B.A., Collier, K.J., and Dixon, J.E. 1984. Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin. Proc. Natl. Acad. Sci. U.S.A. 81:726‐730.
   Eng, J., Du, B.‐H., Pan, Y.‐C.E., Chang, M., Hulmes, J.D., and Yalow, R.S. 1984. Purification and sequencing of a rat intestinal 22 amino acid C‐terminal CCK fragment. Peptides 5:1203‐1206.
   Harper, A.A. and Raper, H.S. 1943. Pancreozymin:A stimulant of the secretion of pancreatic enzymes in extracts of the small intestine. J. Physiol. (Lond) 102:115‐125.
   Hughes, J., Boden, P., Costall, B., Domeney, A., Kelly, E., Horwell, D.C., Hunter, J.C., Pinnock, R.D., and Woodruff, G.N. 1990. Development of a class of selective cholecystokinin type B receptor antagonist having potent anxiolytic activity. Proc. Natl. Acad. Sci. U.S.A. 87:6728‐6732.
   Ivy, A.C. and Oldberg, E. 1928. A hormone mechanism for gallbladder contraction and evacuation. Am. J. Physiol. 86:599‐613.
   Jensen, R.T., Qian, J.‐M., Lin, J.‐T., Mantey, S.A., Pisegna, J.R., and Wank, S.A. 1994.Distinguishing multiple CCK receptor subtypes. Studies with guinea pig chief cells and transfected human CCK receptors. Ann. N.Y. Acad. Sci. 713:88‐106.
   Jorpes, E. and Mutt, V. 1966. Cholecystokinin and pancreozymin, one single hormone? Acta Physiol. Scand. 66:196‐202.
   Lin, C.W., Holladay, M.W., Witte, D.G., Miller, T.R., Wolfram, C.A.W., Bianchi, B.R., Bennett, M.J., and Nadzan, A.M. 1990. A71378: A CCK agonist with high potency and selectivity for CCK‐A receptors. Am. J. Physiol. 258:G648‐G651.
   Lucaites, V.L., Mendelsohn, L.G., Mason, N.R., and Cohen, M.L., 1991. CCK‐8, CCK‐4 and gastrin induced contraction of guinea pig ileum: Evidence for differential release of acetylcholine and substance P by CCK‐A and CCK‐B receptors. J. Pharmacol. Exp. Ther. 256:695‐703.
   Makovec, F., Bani, M., Cereda, R., Pacini, M.A., Revel, L., Rovati, L.A., Rovati, L.C., and Setmikar, I. 1987. Pharmacological properties of lorglumide as a member of a new class of cholecystokinin antagonists. Arzneim. Forsh. 37(11):1265‐1268.
   Mutt, V. and Jorpes, J.E. 1968. Structure of porcine cholecystokinin‐pancreozymin. Eur. J. Biochem. 6:156‐162.
   Rehfeld, J.F., 1978. Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system. J. Biol. Chem. 253:4022‐4030.
   Trist, D.G., Leff, P., Black, J.W., Gerskowitch, V.P., and Shankley, N.P. 1987. Resultant action of cimetidine in a cardiac adenylate cyclase assay:its elucidation by concentration‐ratio analysis. J. Pharmacol. Exp. Ther. 243:1043‐1047.
   Wank, S.A., Pisegna, J.R., and De Weerth, A. 1994. Cholecystokinin receptor family. Molecular cloning, structure and functional expression in rat, guinea pig and human. Ann. N.Y. Acad. Sci. 713:49‐66.
Key References
   Corsi et al., 1993 See above.
  Describes the method for the guinea pig ileum together with the application of concentration‐ratios analysis.
   De Lean et al., 1978 See above.
  Explanation of simultaneous nonlinear curve fitting.
   Reeve, J.R., Eysselein, V., Solomon, T.E., and Go, V.L.W. (eds.) 1994. Cholecystokinin. Annals New York Academy Sciences. Vol. 713.
  A well balanced review of cholecystokinin, including historical perspectives.
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