Characterization of Cholecystokinin Receptors on Rat Pancreatic Membranes*

Abstract

In the present study cholecystokinin (CCK) binding to its receptors on rat pancreatic membranes was characterized by the use of biologically active radioiodinated CCK₃₃prepared by conjugation of [¹²⁵I]Bolton-Hunter reagent ([¹²⁵I]BH) to CCK_33. At pH 7.4 and 24 C, the binding of 25 pM [¹²⁵I]BHCCK to pancreatic receptors was maximal after 120 min and reversible upon the addition of 1 μM unlabeled ligand. [¹²⁵I]BHCCK binding was also tested in membrane preparations from a variety of tissues, but specific binding was found only in the pancreas and cerebral cortex. Scatchard analysis of the pancreas binding data revealed a single class of CCK-binding sites, with a Kd of 1.35 nM and a binding capacity of 204 fmol/mg protein. Optimal [¹²⁵I]BH-CCK binding required 5 IDM Mg²⁺ and 1 mM EGTA; in contrast, monovalent cations (either Na⁺ or K⁺) inhibited binding. [¹²⁵I]-BH-CCK binding exhibit d an acid pH optimum of 5.5; the increase in binding at this pH was due primarily to an increase in capacity (to 399 fmol/mg protein). CCK₁₃ was 4-fold less potent than CCK8 in inhibiting [¹²⁵I]BHCCK binding, whereas desulfated CCK_8, gastrin I and II, and CCK, were 200- to 30,000-fold less potent than CCK₈ in competing for the CCK receptor. Thus, a total membrane fraction from the prancreas showed a selectivity for CCK analogs Containing the terminal CCK octapeptide sequence and a sulfated tyrosine at the seventh amino acid from the carboxyl-terminus. Dibutyryl cGMP, an inhibitor of CCK binding and action in intact pancreatic acinar cells, also inhibited CCK binding to pancreatic particle receptors; in contrast, neither cGMP nor dibutyryl cAMP was effective in inhibiting [¹²⁵I]BH-CCK binding. The present studies demonstrate, therefore, that in the rat, specific CCK receptors exist in the pancreas and brain, and that in the pancreas, these receptors have a very high degree of selectivity for the CCK molecule.