Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179

Abstract

The full peptide antagonist of the pancreatic cholecystokinin (CCK) receptor, JMV 179, [BocTyr(S0,H)-Ahx-Gly-dTrp-Ahx-Asp phenylethyl ester, where Tyr(S0,H) = sulfated tyrosine, Ahx = 6-aminohexanoic acid] was modified at its N-terminus by incorporation of p-hydroxyphenyl propionate (Bolton-Hunter reagent, BH) and was subsequently radioiodinated. After HPLC purification, lZ51-BH-JMV-179, a CCK antagonist radioligand of high specific activity (2000 CUmmol) was obtained. '251-BH-JMV-179 bound to a single population of sites on rat pancreatic plasma membranes, (K, = 3.9 nM, B,,, = 40 pmoVmg protein). Binding was dependent on time, temperature, and protein concentration, and was fully reversible. JMV 179 radioligand detected four times as many sites This study, with the first CCK peptide antagonist radioligand, demonstrates that CCK receptors exist in two interconvertible affhity states regulated by guanine-nucleotide-binding regulatory protein(s) in rat pancreatic plasma membranes. JMV 179 radioligand does not induce receptor coupling but distinguishes the two affinity states of the CCK receptors. JMV 179 reveals the existence of populations of high-affinity and low-affinity sites for CCK which had not previously been detected by agonist radioligand binding, thus suggesting heterogeneity of CCK receptor sites in membranes.Studies during the last decade have abundantly documented the wide distribution of cholecystokinin (CCK) peptides within the gastrointestinal tract as well as the nervous system [l]. Pancreatic acinar cells from rat possess only Asubtype CCK receptors [2-41, the occupation of which by CCK agonists stimulates enzyme exocytosis [ 5 ] . The recent cloning and sequencing of the CCK receptor cDNA has provided important information [6]. However, many questions

Synthesis and in Vitro Characterization of Radioiodinatable Benzodiazepines Selective for Type 1 and Type 2 Cholecystokinin Receptors

“…All these states were, however, bound with high affinity by an antagonist, resulting in a larger fraction of CCK 1 receptor states with high affinity for the antagonist than for the agonist. Accordingly, radiolabeled CCK 1 receptor antagonists labeled considerably more CCK 1 receptor binding sites in the rat pancreas compared with CCK 1 receptor agonists 31, 32. The present study indicates that the same is true for human CCK 1 receptors expressed in tumors.…”

Development of a photoreactive parathyroid hormone antagonist to probe antagonist–receptor bimolecular interaction

“…Therefore, the receptor‐binding affinity of an agonist measured as competition of an agonist‐based radioligand would be higher than that measured with an antagonist‐based radioligand, whereas antagonists should display comparable affinities, as was observed in our study. Similar observations were reported from binding studies on the rat pancreatic CCK‐A receptor using a CCK antagonist radioligand (41).…”

Molecular cloning, expression and pharmacological characterization of the canine cholecystokinin 1 receptor

“…In addition to the biphasic nature of the saturation binding isotherm, it was also observed that the slope of the competition–inhibition curve for L‐364,718 was significantly greater than unity in the assays of both the canine and human CCK1 receptor. This finding is not unique as steep Hill slopes have been previously reported for L‐364,718 in rat pancreatic tissue (Silvente Poirot et al ., 1993; n H =2.01, p K I =9). Interestingly, the slope for L‐364,718 was not steep when measured at the canine CCK2 receptor although, due to the 10‐fold lower affinity for this receptor, the competition–inhibition curve was obtained over a higher concentration range.…”