Characterization of Cholecystokinin (CCK) Receptors

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Cholecystokinin (CCK) and gastrin are structurally related peptide hormones synthesized and secreted by endocrine cells in the gastric antrum, the proximal small intestine, and the central and enteric nervous systems. A broad spectrum of cells express receptors and represent physiologic targets for these peptides. The major physiologic targets for gastrin in the oxyntic gastric mucosa, including the parietal cells and select enterochromaffin cells, while CCK has a broader range of targets, with most involved in nutrient assimilation and appetite control, and the regulation of anxiety. This unit describes binding assays for the determination of receptor number, ligand affinity, and inhibition constants for both CCK_A and CCK_B receptors, in addition to procedures detailing the preparation of tissues from various sources.

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Basic Protocol 1: Binding Assays for CCK1 and CCK2 Receptors
Support Protocol 1: Preparation of Dispersed Rat Pancreatic Acini
Support Protocol 2: Preparation of Enriched Plasma Membranes from Rat Pancreas
Reagents and Solutions
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Binding Assays for CCK1 and CCK2 Receptors

Materials

Skatron buffers: 0.15 M NaCl and 0.2% (w/v) BSA
Radioligand (select one or more, according to experimental design):
[¹²⁵ I]Bolton‐Hunter‐CCK‐8 (full agonist at CCK₁ and CCK₂ receptors; 2200 Ci/mmol, NEN Life Sciences; preferred radioligand for this procedure)
[¹²⁵ I‐Tyr¹² ]human gastrin‐I (high‐affinity radiolabeled CCK₂ receptor agonist with low affinity for the CCK₁ receptor; 2200 Ci/mmol, NEN Life Sciences)
[N ‐methyl‐³ H]L‐364,718 (selective CCK₁ receptor antagonist; 60 to 87 Ci/mmol, NEN Life Sciences)
[3‐methylphenyl‐2,4,6‐³ H]L‐365,260 (selective CCK₂ receptor antagonist; 70‐100 Ci/mmol, NEN Life Sciences)
[³ H]PD‐140376 (selective CCK₂ receptor antagonist; 40‐60 Ci/mmol, Amersham)
Krebs Ringer's/HEPES (KRH) medium (see recipe ), ice‐cold
Competing unlabeled receptor ligands (select one or more, according to experimental design; prepare all stock solutions at 0.05 mM):
0.05 mM CCK‐8 stock solution (see recipe )
CCK‐8‐desulfate (CCK‐8‐DS; Bachem)
CCK‐4 (Research Plus)
Gastrin 17‐I (Bachem)
Gastrin 17‐II, sulfated (Bachem)
L‐364,718 (Dr. Roger Freidinger; Merck Research Laboratories)
L‐365,260 (Merck Research Laboratories.)
Target cells (e.g., a receptor‐bearing cell population; see protocol 2 ) or a membrane preparation from those cells (see protocol 3 )
12 × 75–mm polypropylene test tubes (Sarstedt)
Cell harvester (Skatron)
Receptor‐binding filtermats (Skatron)
Software for analysis of binding data (e.g., LIGAND; Munson and Rodbard, and unit 1.3 )

Support Protocol 1: Preparation of Dispersed Rat Pancreatic Acini

Materials

100‐ to 125‐g male Harlan/Sprague‐Dawley rat
Krebs Ringer's/HEPES (KRH) medium (see recipe ), ice cold
1 U/µl collagenase (Worthington Biochemical) in recipeKRH medium
Source of oxygen

OMG dissecting instruments
Paraffin‐coated petri dish (fill with melted paraffin and allow to set)
6‐ml syringe with 27‐G needle
Silanized 50‐ml Erlenmeyer flask
No. 200‐mesh nylon

Additional reagents and equipment for counting cells (Phelan, )

Support Protocol 2: Preparation of Enriched Plasma Membranes from Rat Pancreas

Materials

Twenty‐five 125‐ to 150‐g male Harlan/Sprague‐Dawley rats
0.3 M and 2 M sucrose solutions (see recipe ), ice cold
Krebs Ringer's/HEPES (KRH) medium (see recipe ), ice cold

Wheaton Dounce homogenizers: 40‐ml equipped with “A” (tight) and “B” (loose) pestles, and 8‐ml equipped with “A” pestle
Beckman ultracentrifuge with SW28 rotor and 38‐ml polyallomer ultracentrifuge tubes, plus Ti50.2 rotor and 26.3‐ml polycarbonate bottles with caps (or equivalent)
Cheesecloth, 2 layers

Additional reagents and equipment for dissecting and excising pancreases (see protocol 2 ) and for determining protein concentration ( appendix 3A )

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Figures

Figure 1.12.1 Amino acid sequences of human (A ) CCK and (B ) gastrin. pGlu, pyroglutamic acid.

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Figure 1.12.2 Typical displacement curves for saturable binding of a CCK‐like radioligand to membrane preparations from Chinese hamster ovary cell lines expressing CCK₁ and CCK₂ receptors. Displacement by various unlabeled peptides in the CCK/gastrin family is shown.

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Videos

Literature Cited

Literature Cited
	Beinborn, M., Lee, Y.‐M., McBride, E.W., Quinn, S.M., and Kopin, A.S. 1993. A single amino acid of the cholecystokinin‐B/gastrin receptor determines specificity for non‐peptide antagonists. Nature 362:348‐350.
	Freidinger, R.M. 1989. Cholecystokinin and gastrin antagonists. Med. Res. Rev. 9:271‐290.
	Gaisano, H.Y., Klueppelberg, U.G., Pinon, D.I., Pfenning, M.A., Powers, S.P., and Miller, L.J. 1989. Novel tool for the study of cholecystokinin‐stimulated pancreatic enzyme secretion. J. Clin. Invest. 83:321‐325.
	Johnson, L.R. 1985. Gastrin receptor assay. Methods Enzymol. 109:56‐64.
	Lee, Y.‐M., Beinborn, M., McBride, E.W., Lu, M., Kolakowski, L.F., and Kopin, A.S. 1993. The human brain cholecystokinin‐B/gastrin receptor: Cloning and characterization. J. Biol. Chem. 268:8164‐8169.
	Miller, L.J., Rosenzweig, S.A., and Jamieson, J.D. 1981. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor, Nα(125I‐desaminotyrosyl)CCK‐8, and its interactions with pancreatic acini. J. Biol. Chem. 256:12417‐12423.
	Munson, P.J. and Rodbard, D. 1980. LIGAND: a versatile computerized approach for characterization of ligand‐binding systems. Anal. Biochem. 107:220‐239.
	Phelan, M.C. 1998. Techniques for mammalian cell tissue culture. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3F.1‐A.3F.14. John Wiley Sons, New York.
	Powers, S.P., Pinon, D.I., and Miller, L.J. 1988. Use of N,O‐bis‐Fmoc‐D‐Tyr‐ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides. Int. J. Pept. Protein Res. 31:429‐434.
Key References
	Miller et al. 1981. See above.
	Detailed description of preparation of ideal agonist radioligand for both types of CCK receptors, and its application to characterize CCK1 receptors on rat pancreatic acinar cells.
	Gaisano et al. 1989. See above.
	Comparison of different types of radioligands and of use of intact cells versus membranes for binding assays.
	Freidinger. 1989. See above.
	Excellent review of antagonists for both type 1 and type 2 CCK receptors.

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Characterization of Cholecystokinin (CCK) Receptors

Abstract

Table of Contents

Materials

Basic Protocol 1: Binding Assays for CCK1 and CCK2 Receptors

Support Protocol 1: Preparation of Dispersed Rat Pancreatic Acini

Support Protocol 2: Preparation of Enriched Plasma Membranes from Rat Pancreas

Figures

Videos

Literature Cited