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J. Biol. Chem., Vol. 275, Issue 25, 19115-19120, June 23, 2000

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Species Differences between Rat and Mouse CCK_A Receptors Determine the Divergent Acinar Cell Response to the Cholecystokinin Analog JMV-180^*

Baoan Ji, Alan S. Kopin^§, and Craig D. Logsdon^¶

From the  Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622 and ^§ Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111

Received for publication, March 1, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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_AR) was used to express the rat receptor in acini from CCK_A receptor-deficient mice (CCK_AR /). Infection of CCK_AR / acini in vitro with pAdCCK_AR led to a time-dependent increase in ¹²⁵I-CCK₈ 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_AR 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_AR / mouse acini infected with AdCCK_AR 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 acinar cell secretion, an oscillatory pattern of intracellular Ca²⁺ concentration, stimulation of protein synthesis, and little measurable increase in either inositol 1,4,5-trisphosphate or diacylgycerol. In contrast, high concentrations of full agonists cause an inhibition of acinar cell secretion, a peak and plateau pattern of intracellular Ca²⁺ concentration, inhibition of protein synthesis, and large increases in inositol 1,4,5-trisphosphate and diacylgycerol. In mice, JMV-180, like CCK-8, is a full agonist and shows a similar pattern of biphasic responses (5). In contrast, when administered to rats, JMV-180 lacks the biphasic response and only generates the pattern observed with low concentrations of CCK-8 (5, 8).

The explanation for this interspecies variability is not clear. There are 23 amino acid differences between the rat and mouse CCK_A receptor sequences. Therefore, one possibility is that interspecies polymorphisms in the CCK_A receptor structure account for the observed functional variability in the action of JMV-180. An alternative explanation for the observed species dependence of JMV-180 signaling is that there are significant differences between acinar cells in rats and mice. A recent study supporting the latter hypothesis showed that there were no major differences between the binding or coupling of rat and mouse CCK_A receptors when the receptors were expressed in CHO cells (4). However, CHO cells are fibroblastic cells derived from the Chinese hamster ovary and may not accurately reflect the cellular context of a pancreatic acinar cell.

The initial goal of the current study was to express a functional rat CCK_A receptor in a mouse acinar cell and examine the responses to CCK-8 and JMV-180 in order to determine whether the physiologic cellular context influences the observed differences in agonist activity. We utilized adenoviral-mediated gene transfer to express rat CCK_A receptors in pancreatic acini, because we have previously found this to be a highly efficient approach (9). To avoid complications due to the presence of endogenous mouse CCK_A receptors, we utilized acini prepared from CCK_A receptor-deficient mice (CCK_AR /) (10). We found that adenoviral infection allowed expression of ectopic receptors to physiologic levels in nearly 100% of the cells within a few hours. At the titers utilized in this study, adenovirally infected acini secreted amylase and synthesized protein identically to uninfected acini. Using this approach we observed that ectopically expressed rat CCK_A receptors mimicked the pattern of biological responses (i.e. secretion and protein synthesis) to CCK-8 and JMV-180 that is found in studies of wild-type rat acini. These results contrast with what was observed with endogenous mouse CCK_A receptors. Taken together, these data indicate that CCK_A receptor structural differences account for the divergence in JMV-180 activity in these two species and, furthermore, illustrate the importance of investigating receptor function in the context of physiologically relevant cell models.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Harlan Sprague-Dawley rats were obtained from Harlan Inc. (Indianapolis, IN). CCK_AR / mice were generated as described previously (11). 129/SVIMJ control mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). Collagenase (CLSPA grade) was purchased from Worthington Biochemicals (Freehold, NJ). Sulfated CCK-8 was obtained from Bachem Bioscience Inc. (Torrance, CA). Amylase 3 reagent was obtained from Sigma. Protein assay reagent was obtained from Bio-Rad. [³H]Leucine was purchased from Amersham Pharmacia Biotech. Trichloroacetic acid was from J. T. Baker Inc. [¹²⁵I-Bolton-Houter]CCK₈ (81,400 GBq/mm) was obtained from NEN Life Science Products. All other materials were purchased from Sigma.

Construction of a Recombinant Adenovirus Encoding the Rat CCK_A Receptor-- A recombinant adenovirus encoding the rat CCK_A receptor was produced according to the method of He et al. (12). Briefly, a HindIII/BamHI fragment that encoded the full-length rat CCK_A receptor (13) (kindly provided by S. A. Wank, National Institutes of Health, Bethesda, MD) was blunted with Klenow and cloned into the EcoRV site of the pAd-Track-CMV shuttle vector, producing pAd-Track-CMV-CCK_AR plasmid. Under the control of distinct CMV promoters, this plasmid expresses the rat CCK_A receptor and, in parallel, green fluorescent protein (GFP). The construct was linearized with PmeI and cotransfected together with the adenoviral backbone vector, pAd-Easy-1, into Escherichia coli strain BJ5183. Recombinant cosmids were selected with kanamycin and screened by BamHI and PacI digestion. The adenoviral construct was then cleaved with PacI and transfected in a packaging cell line (human embryonic kidney 293 cells). An adenovirus (AdLacZ) expressing bacterial -galactosidase and GFP, each under the control of a separate CMV promoter, was a gift from Dr. He (John Hopkins Oncology Center, Baltimore, MD) and utilized as a control. The recombinant virus was concentrated using a CsCl gradient. The titer of the viral stocks was estimated based on the density of GFP-expressing cells. Virus was stored in 10% glycerol at 80 °C in aliquots of 50 µl.

Preparation of Acini and Infection with the Virus-- Acini were prepared by methods previously described (14). In brief, the pancreas was excised from freely fed adult male Harlan Sprague-Dawley rats or 129/SVIMJ mice. Acini were prepared by enzymatic digestion with purified collagenase, followed by mechanical shearing. Acini were then filtered through 150-µm Nitex mesh, purified by sedimentation through 4% bovine serum albumin in Dulbecco's modified Eagle's medium. The acini were suspended in HEPES-Ringer buffer (HR) containing 1% bovine serum albumin, 0.1 mg/ml soybean trypsin inhibitor, and 127 mM NaCl, 4.7 mM KCl, 1.06 mM MgCl₂, 1.28 mM CaCl₂, 1.0 mM Na₂HPO₄, 10 mM HEPES, 2 mM L-glutamine, and 10 mM D-glucose and essential amino acids. The pH was adjusted to 7.4 and equilibrated with 100% O₂ before use. The acini of CCK_AR / mice were infected with virus encoding the rat CCK_AR. Acini of wild-type mice and rats were infected with virus encoding -galactosidase. All virus was used at ~10⁷ plaque-forming unit/mg of protein. Virus was added to acini in 3 ml of HR buffer for 10 min. The acini were then diluted with HR to 13 ml, transferred to 100-mm dishes, and incubated at 37 °C in a humidified 5% CO₂ atmosphere.

JMV-180 Competition Binding Assays-- The affinity of JMV-180 for acini expressing CCK_ARs was assessed by competition binding experiments using 5 pM ¹²⁵I-CCK-8 as radioligand. All experiments were done in HR buffer with the bovine serum albumin concentration adjusted to 0.1% and inclusion of 0.5 mg/ml bacitracin. Acini were incubated in polystyrene tubes at 37 °C with increasing amounts of nonradioactive JMV-180 for 90 min; preliminary studies showed that binding reached a steady-state by 90 min. Nonspecific binding was assessed in the presence of 100 nM unlabeled CCK-8. Acini were separated from unbound CCK by centrifugation at 300 × g for 3 min and then washed twice with ice-cold 0.9% NaCl. The cells were then solubilized with 1 ml of 0.1 N NaOH, and radioactivity was quantified in a -counter. Protein content was determined on samples after counting. Binding affinity was calculated from JMV-180 competitive binding experiments using the Graphpad Prism 3.0 program (Graphpad Software Inc, San Diego, CA).

Analysis of CCK and JMV-180 Induced Increases in Intracellular Ca²⁺ Concentration-- Analysis of intracellular Ca²⁺ concentration was conducted using ratiometric imaging of Fura-2-loaded cells as described previously (15). In brief, acini were incubated with 5 µM fura 2-AM at 37 °C for 30 min and then washed and resuspended in HR buffer. Fura-loaded acini were transferred to a closed chamber, mounted on the stage of a Zeiss Axiovert inverted microscope, and continuously superfused at 1 ml/min with HR buffer and different concentrations of CCK-8 or JMV-180 at 37 °C. Measurement of emitted fluorescence and calibration of these signals to yield a measurement of intracellular Ca²⁺ were performed using an Attofluor digital imaging system (Rockville, MD), as described previously (15).

Analysis of Amylase Secretion-- Amylase secretion in response to CCK-8 or JMV-180 was measured as described previously (9). Briefly, acini were washed and resuspended in HR at ~1 mg/ml. Aliquots of 3.5 ml were distributed into 25-ml polycarbonate Erlenmeyer flasks and incubated with increasing concentrations of CCK-8 or JMV-180 at 37 °C for 30 min. Incubation was terminated by centrifugation of 1-ml aliquots for 15 s at 15,000 × g. The concentration of amylase in the medium was measured using the Amylase 3 reagent. Results were expressed as a percentage of initial acinar amylase content.

Incorporation of Amino Acid into Protein-- The effects of CCK-8 and JMV-180 on protein synthesis were examined using [³H]leucine as described previously with minor modifications (16). Briefly, 1-ml aliquots of acinar suspension (1 mg of total protein) were preincubated at 37 °C for 30 min with either CCK-8 or JMV-180 using duplicate flasks for each concentration studied. [³H]Leucine (2 µCi/ml) was then added, and incorporation was terminated 20 min later by diluting with an equal volume of ice-cold 0.9% NaCl containing 20 mM unlabeled leucine. After centrifugation, the sediments were washed with 4 °C saline and disrupted by sonication in 0.75 ml of distilled water. The protein was then precipitated with equal volumes of ice-cold 20% trichloroacetic acid. After 30 min, the trichloroacetic acid precipitates were washed twice with ice-cold 10% trichloroacetic acid and dissolved in 400 µl 0.1 N NaOH. Aliquots were removed for protein assay, and the remaining samples were measured in a liquid scintillation counter. Data for each experimental condition were normalized to the percentage of [³H]leucine incorporation in control acini ([³H]leucine incorporated/total [³H]leucine added/mg of protein × 100).

Statistical Analysis-- Statistical analysis was carried out using Graphpad Prism 3.0 program (Graphpad Software Inc, San Diego, CA). Differences between individual conditions and controls were tested with analysis of variance and the Newman-Keuls multiple range test. Two-way analysis of variance was used to analyze the experiments that had a factorial design. In the analysis, differences were considered significant when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adenoviral-mediated Expression of a Rat CCK_A Receptor-- CCK_A receptors are expressed in the exocrine pancreas of both rats and mice. Species differences in the structure of these receptors are relatively minor, with only 23 amino acids that diverge between respective CCK_AR homologs (Fig. 1). The major structural difference is a seven-amino acid insertion found in the third intracellular loop of the mouse CCK_A receptor. Dissimilarities have previously been noted between the functional effects of the CCK analog JMV-180 in these two species (5, 6). To determine whether these divergent biological responses were the result of variability in the structure of the receptors or due to differences in cellular environment, we utilized an adenoviral construct encoding the rat CCK_A receptor (AdCCK_AR). This virus also expresses GFP under the control of a distinct CMV promoter. In initial experiments, efficiency of the infecting virus was assessed by monitoring GFP levels in acini by fluorescence microscopy. It was observed that adenoviral-mediated gene expression was titer-dependent (data not shown). Using a titer of ~10⁷ plaque-forming unit/mg of acinar protein, nearly 100% of the acini expressed GFP (Fig. 2). Higher titers did not further improve efficiency (data not shown).

View larger version (85K): [in this window] [in a new window]   Fig. 1.   Comparison of mouse and rat CCKA receptor structure. Boxes indicate residues that differ. Dark lines above the sequences indicate predicted locations of transmembrane domains. CCKA receptors from these two species differ by 23 amino acid residues. The major difference is a seven-amino acid insert in the third cytoplasmic loop of the mouse receptor. The mouse and rat sequences shown are from GenBankTM accession numbers AF015963 and M88096, respectively.

View larger version (71K): [in this window] [in a new window]   Fig. 2.   Adenoviral infection of pancreatic acini is highly efficient. Acini were prepared and were infected with 107 plaque-forming units of pAdCCKAR/mg of acinar protein. This adenovirus expresses the rat CCKA receptor and GFP under the control of separate CMV promoters. After a 16-h incubation, acini were observed with fluorescent microscopy and photographed with a digital camera. Acini expressing virally delivered GFP display a green fluorescence. The same field of acini was then photographed under bright-field conditions for comparison. Results show that nearly 100% acini are infected.

Adenoviral-mediated gene expression was also time-dependent. To define the level of receptor expression, ¹²⁵I-CCK binding was assessed at different times after infection with AdCCK_AR. Acini isolated from CCK_AR / mice showed no CCK binding before infection. After infection of CCK_AR / acini with AdCCK_AR, a time-dependent increase in ¹²⁵I-CCK binding was observed (Fig. 3). Specific binding levels comparable with those of acini from CCK_AR +/+ mice were observed ~4 h after infection. Maximal binding levels ~5-fold over control were observed after 16 h. Therefore, all biological responses were assessed 4 h post-infection.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Time-course of adenoviral-mediated CCKAR gene expression in pancreatic acini. Specific binding of 125I-CCK was compared between acini prepared from wild-type mice (black) and infected with pAdLacZ and those from CCKAR / mice infected with pAdCCKAR for various times (striped). Binding was conducted using 125I-CCK (5 pM) with or without excess unlabeled CCK-8 (100 nM) for an additional 90 min at 37 °C. Acini were then washed, and the amount of displaceable binding was determined. The rat CCKA receptor expression levels in infected acini from CCKAR / acini increased over time. Binding levels similar to those observed in acini from control CCKAR +/+ mice were obtained within 4 h after infection. Data shown are expressed as the percentage of binding observed in acini from wild-type mice (11 ± 0.5% total/mg of protein, n = 4) and are means ± S.E. from 4-6 separate experiments.

Binding and Signaling of the Rat CCK_A Receptor Expressed in Mouse Acini-- To determine the affinity of the rat CCK_A receptor expressed in the mouse acinar cells, competition binding experiments were performed after infection of acini from CCK_AR / mice with AdCCK_AR. When expressed in acini from CCK_AR / mice, the rat receptor showed high affinity displaceable ¹²⁵I-CCK binding that was comparable with affinities observed with either mouse or rat wild-type acini (data not shown). JMV-180 competed for binding of ¹²⁵I-CCK (Fig. 4). The affinity of JMV-180 for the rat receptor expressed in the mouse CCK_AR / acinar cell was not significantly different from the affinity of the native mouse receptor assessed in acini from CCK_AR +/+ mice (K_i = 1.4 ± 0.1 versus 1.5 ± 0.39 nM, n = 4, p > 0.05). Infection with control adenovirus had no effect on receptor binding (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Characteristics of JMV-180 binding to mouse pancreatic acini expressing native mouse or ectopic rat CCKA receptors. Competitive binding assays were conducted with acini either from wild-type control mice infected with pAdLacZ (squares) or from CCKA receptor-deficient mice infected with pAdCCKAR (triangles). Four hours after infection acini were incubated with 125I-CCK (5 pM) in the presence of indicated concentrations of JMV-180 for 90 min at 37 °C. Cells were then washed, and radioactivity associated with the cells was assessed. Nonspecific binding was determined using 100 nM CCK-8. Data are expressed as % total binding/mg of protein and are means ± S.E. from four independent experiments.

To characterize the coupling of the rat CCK_AR to calcium-mediated signaling when expressed in mouse acini, cells were loaded with Fura-2AM, and intracellular Ca²⁺ levels were determined by ratiometric fluorescence imaging. CCK_AR / acini without virus infection did not respond to CCK-8 (Fig. 5A) or JMV-180 (data not shown); however, they did show a normal response to stimulation with bombesin. Acini from CCK_AR / mice infected with AdCCK_AR showed an oscillatory pattern of Ca²⁺ increase after stimulation with a low concentration and a peak and plateau type of response when stimulated with a high concentration of CCK-8 (Fig. 5B). The response of AdCCK_AR-infected acini to CCK-8 was similar to the pattern previously observed in acini prepared from CCK_AR +/+ mice or from rats (5). Responses to JMV-180 were not markedly different between acini prepared from wild-type rats and mice. Both showed oscillatory Ca²⁺ responses (Fig. 5, C and E). However, subtle differences were noted. In mice, the oscillations did not return to base line, whereas in rats the oscillations did return to base-line levels. Ca²⁺ responses to JMV-180 of acini from CCK_AR / mice infected with AdCCK_AR resembled the oscillatory activity observe with wild-type rat acini, i.e. with a full return to base line between spikes. It should be noted that these differences in Ca²⁺ signaling were somewhat variable and did not allow a clear distinction between species.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Rat CCKA receptors couple with increases in intracellular Ca2+ in mouse acinar cells. Acini were infected with adenovirus for 4 h and then loaded with Fura 2-AM for 30 min before ratiometric imaging was performed. A, acini from CCKA / mice were infected with pAdLacZ and then stimulated with either CCK-8 (1 nM) or bombesin (Bn, 1 nM) for the time indicated. B, acini from CCKA / mice were infected with pAdCCKAR and then stimulated with either a low (10 pM) or a high (100 pM) concentration of CCK-8 as indicated. C, control mouse acini were infected with pAdLacZ and then stimulated with a maximal concentration of JMV-180 (1 µM). D, acini from CCKA / mice were infected with pAdCCKAR and then stimulated with JMV-180 (1 µM). E, acini from a rat were infected with pAdLacZ and then stimulated with JMV-180 (1 µM). Shown are representative traces from at least 25 acinar cells from one of at least 4 independent experiments.

Biological Responses to Activation of Rat CCK_A Receptors Expressed in Mouse Acini-- To assess the biological activity of mouse acini expressing recombinant rat CCK_A receptors we analyzed the effect of receptor activation on both amylase secretion and protein synthesis. In initial experiments, the effect of adenoviral infection per se on pancreatic acinar cell amylase secretion was determined. Infection of acini isolated from CCK_AR +/+ mice with a control GFP-expressing virus had no significant effect on basal (1.0 ± 0.4 versus 1.8 ± 0.4% total/30 min, n = 3, p > 0.05) or maximal (5.2 ± 1.9 versus 5.1 ± 1.1% total/30 min, n = 3, p > 0.05) CCK-8 stimulated amylase release. Next, the concentration-dependent release of amylase stimulated with either CCK-8 or JMV-180 was assessed. Acini from CCK_AR / mice were compared with acini from control mice and control rats (Fig. 6). In both rat and wild-type mice acini, a typical biphasic dose-response curve for CCK-8 stimulation of amylase release was observed. The percentage of total amylase released in 30 min was less in mice (Fig. 6A) than in rats (Fig. 6C), as has been reported previously (17). In both species, concentrations of CCK-8 above 1 nM caused a reduction in amylase release. By contrast, markedly different patterns of response to stimulation with JMV-180 were observed in rats versus CCK_A +/+ mice. In CCK_A +/+ mice, JMV-180 caused a biphasic stimulation of amylase release, with prominent inhibition observed at supramaximal concentrations (Fig. 6A). In rats, JMV-180 showed monophasic concentration dependence without causing any decrease of amylase release even at supramaximal concentrations (Fig. 6C). In acini from CCK_AR / mice infected with AdCCK_AR the amylase response pattern to CCK-8 was biphasic, similar to that observed in acini from control rats and mice. The level of amylase released was comparable with what was observed in native mouse acini. However, in mouse acini expressing the rat CCK_A receptor, the pattern of amylase release stimulated by JMV-180 was similar to that observed in control rats (monophasic) rather than control mice (biphasic). Although JMV-180 stimulated amylase secretion at low concentrations in rat AdCCK_AR-infected mouse acini, no inhibition of secretion was observed even at concentrations up to 1 µM (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Species differences in secretory responses to JMV-180 are determined by the receptor structure. Acini from control mice, CCKA receptor knockout mice, or rats were infected with virus. Amylase secretion in response to increasing concentrations of CCK-8 and JMV-180 was measured at 4 h. A, acini from control mice infected with the pAdLacZ control virus. Both CCK-8 and JMV-180 had biphasic effects on amylase release. B) acini from CCKA receptor / mice infected with pAdCCKAR. CCK-8 stimulated a biphasic effect on amylase release, but JMV-180 did not show high dose inhibition. C) acini from control rats infected with pAdLacZ control virus. CCK-8 stimulated a biphasic effect on amylase release, but JMV-180 did not show high dose inhibition. Results are means ± S.E. for 3-5 experiments.

To evaluate the effect of receptor activation on protein synthesis, the incorporation of [³H]leucine into protein was analyzed. Differences were noted in the effects of both CCK-8 and JMV-180 on protein synthesis in rats versus mice. In control mice, CCK-8 caused an inhibition of protein synthesis that was significant at a concentration of 0.1 nM (Fig. 7A). In rat acini, CCK-8 inhibited protein synthesis, but these effects required CCK-8 concentrations above 1 nM (Fig. 7C). In control mice, JMV-180 caused a decrease in protein synthesis (Fig. 7A). In contrast, in rat acini, JMV-180 did not inhibit protein synthesis even at concentrations up to 1 µM (Fig. 7C). The pattern of effects of CCK-8 and JMV-180 on protein synthesis in acini isolated from CCK_A / mice infected with AdCCK_AR resembled the observed pattern in the rat. In mouse acini expressing ectopic rat CCK_A receptors, CCK-8 did not significantly inhibit protein synthesis at concentrations below 1 nM (Fig. 7B). In addition, as in rat acini, JMV-180 did not inhibit protein synthesis at any concentration. Overall, the mouse CCK_AR / acini expressing rat CCK_ARs biologically resembled wild-type rat rather than wild-type mouse acini.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Species differences in protein synthesis responses to JMV-180 are determined by CCKA receptor structure. Acini from control mice, CCKA receptor / mice, or rats were infected with virus. Protein synthesis in response to increasing concentrations of CCK-8 and JMV-180 was assessed at 4 h. A, acini prepared from control mice were infected with pAdLacZ control virus. Both CCK-8 and JMV-180 inhibited protein synthesis. B, acini from CCKA receptor / mice infected with pAdCCKAR. CCK-8 caused prominent inhibition at concentrations of 1 nM or above. JMV-180 did not inhibit protein synthesis at any concentration tested. C, acini prepared from rats were infected with pAdLacZ. CCK-8 inhibited protein synthesis at concentrations of 3 nM or above. JMV-180 did not inhibit protein synthesis and caused a small increase in synthesis at the highest concentrations. Results are means ± S.E. for 3-5 experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we expressed rat CCK_A receptors in the context of the mouse pancreatic acinar cell. Although the mouse CCK_A receptor and the rat CCK_A receptor are structurally highly homologous, several important differences in ligand-induced activity were observed. Activation of the mouse CCK_A receptor with the CCK-8 analog JMV-180 had a biphasic effect on secretion, as has been previously noted (5). However, activation of the rat CCK_A receptor in the context of the mouse acini did not show high dose inhibition. This pattern of response is similar to what is observed in wild-type rat acinar cells (5). Furthermore, activation of the mouse CCK_A receptor with JMV-180 inhibited protein synthesis, as has been previously described (5). In contrast, no inhibitory effect was noted when the rat CCK_A receptor was activated in the context of the mouse acinar cell. Thus, the rat CCK_A receptor expressed in the context of the mouse acinar cell responds similarly to what is observed in wild-type rat acini and contrasts with the CCK_AR-induced effects observed in wild-type mouse acini. Therefore, structural differences in the CCK_AR protein are the most likely explanation for the observed divergence in effects of JMV-180 between the two species.

This study utilized adenoviral-mediated gene transfer to express receptors in pancreatic acini. Adenoviral infection has been shown to be a highly efficient means of gene transfer to this cell type (9, 14, 18). In the current study using GFP expression to monitor gene transfer, we observed nearly 100% efficiency. This high level was confirmed in experiments imaging Ca²⁺ release in individual acinar cells, where every cell infected with receptor bearing adenovirus showed a response to CCK-8. Adenoviral infection at the titers used in the current study did not perturb receptor binding, Ca²⁺ signaling, secretion, or protein synthesis. However, previously we noted that adenoviral infection induces an increase in NF-B activation (14). Furthermore, at titers higher than those utilized in the current study, significant deleterious effects were noted (data not shown). Therefore, adenoviral titer is a critical parameter in this type of study. To avoid the complications of altering viral titers we utilized time as a means of controlling receptor expression levels. Receptor levels were observed to increase rapidly after infection, and levels that approximated those found in control acini were observed within 4 h. The biological response observed with adenovirus-mediated receptor expression approximated the biology observed in control cells, illustrating the power of this approach for the study of receptor function.

CCK-8 stimulates an identical pattern of response in both species as well as in mouse acini recombinant rat CCK_A receptors. In both species, the CCK_A receptor is thought to mediate its signaling through interactions with heterotrimeric G proteins of the G_q family (19, 20). An interesting aspect of CCK receptor activation is the dependence on agonist concentration for coupling to separate intracellular signaling events that in turn lead to different biological outcomes (21). At low physiologic concentrations, CCK-8 stimulates an oscillatory Ca²⁺ response (22), stimulates secretion and protein synthesis (21), and increases Erk activity (23). However, at high concentrations, CCK-8 inhibits secretion and protein synthesis (5), disrupts the cytoskeleton (24), activates stress responses including activation of Jun kinase (25) and NFB (14), and induces an acute edematous pancreatitis (26). Thus, the nature of the signaling pathways that differentiate between the effects of high and low concentrations of CCK is potentially of great significance.

The JMV-180 analog is a useful tool in the investigation of CCK receptor signaling. JMV-180 is a synthetic analogue of the COOH-terminal heptapeptide of CCK, with the structure t-butoxycarbonyl-Tyr(SO₃)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester (8) (Nle is norleucine). The most important modification in this CCK analog is in the carboxyl terminus, where aspartate is linked by an ester bond to phenylamine instead of the peptide bond linkage to the terminal-amidated phenylalanine in CCK. Other modifications in this molecule confer increased stability but likely do not affect biological activity (27). In the rat JMV-180 acts as a partial agonist. Activation of the rat CCK_A receptor with JMV-180 elicits biological responses associated with the low concentrations of CCK but not with high concentrations (7). Therefore, JMV-180 has become a popular tool for differentiating between the effects of low and high concentrations of CCK in the rat. In contrast, in the mouse JMV-180 acts as a full agonist, activating the complete range of signaling mechanisms associated with the CCK_A receptor (5).

One possible explanation for the species difference in the effects of JMV-180 is that mouse and rat pancreatic acinar cells present a different environment to the CCK_A receptor. Little is know about the patterns of expression of proteins important to receptor signaling in these cells. Based on work in other cell types, it is well established that receptor function is influenced by a variety of cellular proteins including heterotrimeric G proteins, G protein-linked receptor kinases, arrestins, and RGS proteins (3). Unfortunately, little is known concerning the identity and qualitative distribution of these regulatory molecules within pancreatic acinar cells. However, acinar cells are highly differentiated and specialized for the synthesis, storage, and secretion of digestive enzymes. In this regard, cells from rats and mice share more similarities with each other than with most other cell types. Activation of receptors in the context of the rat acinar cell caused the release of a greater percentage of total cellular amylase content than did activation of either rat or mouse CCK_A receptors in the acini prepared from mice. This species difference has been previously noted (17). Despite these intracellular differences, activation of the rat CCK_A receptor in the context of the mouse acini gave a pattern of effects identical to what is observed in rat acini. Therefore, fundamental differences in receptor structure rather than the cellular environment per se most likely explain the observed differences in JMV-180 effects in rats versus mice.

No appreciable differences were observed in the effects of JMV-180 when the rat and mouse CCK_A receptors were expressed in CHO cells (4). This suggests that the context of the pancreatic acinar cell is required to unravel functional differences between rat and mouse CCK_A receptors. One limitation of the previous study in CHO cells was that it is not possible to investigate the effects of receptor activation on secretion in this model. The only biological response analyzed in the CHO cells was Ca²⁺ release, and no differences between rat and mouse CCK_A receptor stimulation were noted. However, it is possible that the differences between rats and mice in this parameter are small. We observed only subtle differences between the effects of JMV-180 on Ca²⁺ release in rats and mice. In rats, the response to a supramaximal concentration of JMV-180 was exclusively oscillatory, as has been reported previously (22). In mice, the response was also oscillatory in nature, but at least initially, the oscillations did not return to base line. It is of note that activation of the rat CCK_A receptor in the context of the mouse acinar cell produced a pattern of Ca²⁺ release that was more similar to the rat than to the control mouse, although the observed differences were subtle and difficult to quantify. It is possible that differences in the effects of JMV-180 on rat versus mouse CCK_A receptors on CHO cells might be detectable if other cellular parameters, such as protein synthesis, were measured.

In conclusion, expression of the rat CCK_A receptor in the context of receptor-deficient mouse pancreatic acinar cells indicates that differences in receptor structure contribute to the divergent effects of JMV-180 between species. The pattern of responses to JMV-180 activation of rat CCK_A receptors expressed in the context of the mouse pancreatic acinar cell was entirely compatible with what is observed in wild-type rat pancreatic acinar cells and clearly different than what is observed in wild-type mouse acini. Therefore, these data indicate that differences in CCK_A receptor structure between rats and mice lead to distinctive interactions with JMV-180 and explain the observed species dependent differences in biological activity.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK52067 and DK46767 (to A. S. K.) and University of Michigan Gastrointestinal Peptide Center Grant DK34933.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Physiology, Box 0622, University of Michigan, 7710 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622. Tel.: 734-763-2539; Fax: 734-936-8813; E-mail: clogsdon@umich.edu.

Published, JBC Papers in Press, March 24, 2000, DOI 10.1074/jbc.M001685200

	ABBREVIATIONS

The abbreviations used are: CCK, cholecystokinin; CCKR, CCK receptor; CHO, Chinese hamster ovary; CMV, cytomegalovirus; GFP, green fluorescent protein; HR, HEPES-Ringer buffer.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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