Species Differences between Rat and Mouse CCKAReceptors Determine the Divergent Acinar Cell Response to the Cholecystokinin Analog JMV-180

Abstract

The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCK A receptor (AdCCK A R) was used to express the rat receptor in acini from CCK A receptor-deficient mice (CCK A R ؊/؊). Infection of CCK A R ؊/؊ acini in vitro with pAdCCK A R led to a time-dependent increase in 125 I-CCK 8 binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCK A receptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCK A R JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCK A R ؊/؊ mouse acini infected with AdCCK A R resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCK A receptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180.Gene transfer studies provide a powerful approach to investigate the correlation between receptor structure and function. Manipulation of the cDNA encoding a receptor followed by expression of the mutant protein in a cell model allows detailed analysis of the role of specific receptor domains and individual residues. This approach has been widely utilized and has resulted in important advancement in our understanding of G protein-coupled receptor signaling (1). However, it has become increasingly apparent that the choice of cell model used for gene transfer experiments significantly impacts the receptor properties that are observed (2). Differentiated cells express specific subsets of proteins that interact with G protein-linked receptors; examples include G proteins, arrestins, G protein receptor kinases, and RGS proteins (3). The cell model may therefore exert a major influence on the outcome of receptor structure-function studies.Recently it was suggested that interspecies variation in cellular milieu might explain observed differences between the drug-induced responses of the rat versus the mouse CCK A receptor (4). In both species, CCK A receptors bind the endogenous hormone cholecystokinin (CCK) 1 with high affinity. A similar pattern of cell signals and biological responses is in turn triggered in both rats and mice. In contrast, the CCK analog JMV-180 acts as a partial agonist in the rat and a full agonist in the mouse (5-7). Differences between partial and full CCK agonists are striking in the pancreatic acinar cell. Full agonists (e.g. CCK-8) show marked differences in biological effects at both physiologic and supra-physiologic concentrations. At physiologic concentrations, full agonists cause increases in...

Human Pancreatic Acinar Cells Do Not Respond to Cholecystokinin

“…All experiments were conducted with permission of the University of Michigan Internal Review Board for use of Human Materials. Acini were prepared by a modification of the methods previously described for the preparation of rodent acini (Ji et al 2000(Ji et al & 2001. The acini were suspended in HEPESRinger buffer (HR) containing 1% bovine serum albumin.…”

“…The pH was adjusted to 7.4 and equilibrated with 100% O2 before use. In some experiments the acini were infected with virus encoding the human CCK2 or rat CCK1 receptor essentially as previously described (Ji et al 2000).…”

“…That the acini had low levels of basal secretion and responded to carbachol supported the conclusion that the isolated acini were functionally intact. To distinguish between a lack of response due to insufficient CCK receptors versus a lack of CCK receptor related signaling molecules such as G-proteins, we next expressed human CCK2 receptors in acini using adenoviral mediated gene transfer as described previously (Ji et al 2000). Infected acini were incubated for 4 hr, as this amount of time was previously shown to allow a level of expression of receptors in acini isolated from receptor deficient mice that was equivalent to that observed in the control mice (Ji et al 2000).…”

“…To distinguish between a lack of response due to insufficient CCK receptors versus a lack of CCK receptor related signaling molecules such as G-proteins, we next expressed human CCK2 receptors in acini using adenoviral mediated gene transfer as described previously (Ji et al 2000). Infected acini were incubated for 4 hr, as this amount of time was previously shown to allow a level of expression of receptors in acini isolated from receptor deficient mice that was equivalent to that observed in the control mice (Ji et al 2000). Acini infected with the receptor coding adenovirus responded to CCK-8 with a concentration dependent release of amylase that was similar in magnitude to that stimulated by carbachol ( fig.…”

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

“…As a result, this method offers the major advantage of extending acinar cell viability to 7-10 days in vitro. Furthermore, this method is currently preferred to acinar cell isolation [9][10][11][12] because maintenance of intercellular contacts, including cell coupling by gap junctions, is an essential determinant of the exocrine pancreatic acinar cell phenotype 13 .…”