Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J

Abstract

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors ( K D of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17 I (G-17 I)) over selective receptors (20% with a K D of 1 nM and 1 μM for, respectively, CCK-8 and G-17 I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17 I and CCK-4. G-17 I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2–17) I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17 I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [ 32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two G sα (the 50 kDa form predominating over the 45 kDa form) and one G iα (41 kDa). However, G i and G s may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17 I-dependent amylase secretion.