Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas

Abstract

Cholecystokinin/gastrin receptors in the pancreas of newborn (3-day-old) rats are of type A, as in control mature rats, revealed by pharmacological analysis of specific ' 251-Bolton-Hunter-reagent- day-old rats, 90 -125 kDa in 10-day-old rats and 85 -100 kDa in 14-day-old and 21 -day-old rats, as found in control adult rats. Endo-a-N-acetylglucosaminidase F treatment yielded a core protein of 42 kDa in all developmental stages. These findings are consistent with an age-related postnatal expression of distinct glycoforms of pancreatic cholecystokinin receptors. Furthermore, it was observed that the period 2-3 weeks after birth, characterized by stabilization of the mass of the cholecystokinin receptor, precedes the dramatic increase in the receptor number.Cholecystokinin (CCK) and gastrin are hormonal peptides that share the same C-terminal pentapeptide amino acid sequence [I]. Pancreatic acinar cells from dog and guinea pig have been shown to possess two types of CCK receptor [2 -61. The CCKA receptor type, has a high affinity for CCK, and the pancreatic CCKn or gastrin receptor type has a high affinity for gastrin and poorly discriminates between CCK and gastrin peptides.The existence of specific CCK receptors in term foetus and neonatal rat pancreas was previously documented, although acini or lobules seem to be unresponsive to CCK stimulation near birth [7-91. It was also shown that the capacity of the high-affinity sites on rat pancreatic acini, as well as amylase secretion due to CCK stimulation, gradually increases with

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

“…Indeed, CCK radioligands were found to bind to a single class of sites on plasma membranes having an intermediate affinity between the high-affinity and the low-affinity sites existing on acini [3, 4, 7, 81. In contrast to previous reports, we recently demonstrated by using a CCK agonist radioligand that CCK binding occurred on pancreatic plasma membranes with an affinity as high as that of the high-affinity sites of pancreatic acini [20]. However, low-affinity sites for CCK were not revealed [20]. In the present study, performed on identically prepared membranes, inhibition of 1251-BH-JMV-179 binding by CCK agonist allowed detection of both high-affinity and low-affinity sites for CCK agonists.…”

“…3B). Surprisingly, the binding capacity for T-BH-JMV-179 was fourfold higher than that for the agonist radioligand 1251-BH-[Thr28, Ahx3l]CCK-(25 -33), as previously reported by us [20]. As expected for an antagonist ligand, binding of IZ5I-BH-JMV-179 was insensitive to the presence of 0.1 mM GTP[S].…”

“…The reason lies in the discrepancies observed between CCK binding data obtained with pancreatic acini and with membranes prepared from acini. Indeed, CCK radioligands were found to bind to a single class of sites on plasma membranes having an intermediate affinity between the high-affinity and the low-affinity sites existing on acini [3, 4, 7, 81. In contrast to previous reports, we recently demonstrated by using a CCK agonist radioligand that CCK binding occurred on pancreatic plasma membranes with an affinity as high as that of the high-affinity sites of pancreatic acini [20]. However, low-affinity sites for CCK were not revealed [20].…”

“…In addition, analysis of '2sI-BH-JMV-179 binding inhibition by CCK, together with data from a previous study [20], provide new elements of information concerning the detection by antagonist radioligands such as "'I-BH-JMV 179 (this study) and 3H-labelled L 364 718 [ll], of four or five times as many sites as by agonist radioligands. Indeed, the population of high-affinity sites previously identified by the agonist radioligand represents approximately 10.0 pmol/ mg protein [20] while that labelled by the antagonist radioligand, as presently documented, represents 40 pmol/mg protein.…”

“…Enriched plasma membranes were prepared rigorously as described in a previous paper [20], by homogeneization in 0.3 M sucrose plus protease inhibitors followed by a gradient separation 1.3 M/0.3 M at 150000 X g for 3 h. Membrane protein concentration was determined by the method of Bradford [21] using the Bio-Rad protein assay kit.…”