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The existence of high- and low-affinity cholecystokinin (CCK)-A receptors on rodent pancreatic acini is well established. Until recently, CCK was believed to act directly on pancreatic acini to stimulate pancreatic secretion in both rodents and humans. However, conclusive evidence that human pancreatic acini lack functional CCK-A receptors has been presented. Despite substantial differences in rodent and human pancreatic physiology, CCK appears to act via vagal cholinergic pathways to mediate pancreatic secretion in both species. Structural and functional evidence suggests that CCK acts on vagal afferent fibers, which may explain how CCK doses that produce physiologic plasma CCK levels act via vagal cholinergic pathways to stimulate pancreatic secretion. Although most knowledge of vagal CCK-A receptors comes from research on rodents, physiologic studies suggest that this information is applicable to humans. In contrast to its effect on satiety, which is mediated by low-affinity vagal CCK-A receptors, CCK acts through high-affinity CCK-A receptors to evoke pancreatic secretion, suggesting that different affinity states of the vagal CCK receptors mediate different digestive functions. Vagal afferent pathways also transmit sensory information about the mechanical and physiochemical state of the digestive tract, mediated in part by serotonin, which, in turn, influences pancreatic secretion. A synergistic interaction between CCK and serotonin at the level of the nodose ganglia may explain the robust postprandial pancreatic secretion despite a modest postprandial increase in plasma CCK. Important physiologically, these findings not only explain discrepancies in previous in vivo vs. in vitro studies, but they revolutionize our current concept of the mechanism of CCK on pancreatic exocrine secretion.

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.

The cell model studied has a fundamental influence on the function and regulation of G protein linked receptors. These cell-dependent effects are illustrated in the current communication focusing on M3 muscarinic, CCK and GRP receptors. Receptors interact with multiple cellular mechanisms. The most obvious are those involved in coupling to signaling mechanisms such as G proteins. Receptors are themselves phosphorylated and dephosphorylated by cellular kinases and phosphatases. Receptors may sequester, internalize, down-regulate and recycle via interactions with a number of separate cellular mechanisms. When the number and complexity of interactions between the cell and the receptor are taken into account it is not surprising that the cell model has a primary influence on receptor function and regulation. The implications of the importance of the cell model in receptor function for studies aimed at answering physiologic questions are discussed.

Rat cholecystokinin-B (CCK-B) receptors were transfected into Chinese hamster ovary (CHO)-K1 (CHO-CCK-B) and Swiss 3T3 (Swiss 3T3-CCK-B) cells, and the effects of receptor activation on cell proliferation and intracellular signaling were investigated. CCK octapeptide (CCK-8) treatment had no effect on cell growth in quiescent CHO-CCK-B cells but inhibited DNA synthesis, proliferation, and colony formation when the cells were grown in fetal bovine serum (FBS). In contrast, CCK-8 stimulated DNA synthesis in quiescent Swiss 3T3-CCK-B cells and had no effect when the cells were grown in FBS. These differences in growth responses were not due to differences in the level of receptor expression, as similar numbers of receptors were present in both cell types. To determine whether the different growth effects were due to differences in receptor coupling to common second messenger pathways, we investigated the effects of CCK-8 on several known intracellular signals. In both cell types, CCK-8 stimulated increases in intracellular Ca2+ concentration and polyphosphoinositide hydrolysis with similar potencies and efficacies. CCK-8 also stimulated arachidonate release from both cell types, although the potency was higher in the CHO cells. Adenosine 3',5'-cyclic monophosphate generation was observed at high agonist concentrations in both cell types and was much greater in cells with higher receptor density. In summary, receptor activation had opposite effects on growth parameters in CHO and Swiss 3T3 cells, but only minor differences were observed in the characteristics of CCK-B receptor coupling to specific second messengers in the two cell types. Thus cellular context is a principal determinant of the biological effects of CCK-B receptor activation, and differences in biological responses may occur independently of major differences in receptor coupling.

Background & Aims: Cholecystokinin (CCK) acting via CCK(A) receptors and gastrin acting via CCK(B) receptors exert trophic effects on a variety of nontransformed tissues. However, their role as hormonal regulators of pancreatic cancer is controversial. The aim of this study was to determine the effects of activation of CCK(A) and CCK(B) receptors on the growth of human pancreatic cancer cells in vitro., Methods: Two human pancreatic cell lines MiaPaca-2 and Panc-1 were transfected stably with both CCK receptor subtypes. Effects of CCK on various growth parameters including DNA synthesis, nuclear labeling, and colony formation were evaluated., Results: Cells expressing either receptor subtype, but not untransfected cells, bound ligand and mobilized Ca2+ in response to CCK. CCK treatment caused a sustained pronounced inhibition of anchorage-independent growth. Similarly, CCK treatment inhibited anchorage-dependent growth. Receptor activation caused a concentration and time-dependent reduction in [3H]thymidine incorporation and nuclear labeling in cells cultured anchored to a plastic substrate. However, these effects on anchorage-dependent growth were transient, suggesting cellular desensitization., Conclusions: These data indicate that both CCK receptor subtypes can mediate growth inhibitory responses in pancreatic cancer cell lines and raise the possibility that CCK exerts a predominant growth inhibitory action on human pancreatic cancer cells.

It was recently found that cholecystokinin (CCK) activates mitogen-activated protein kinases (MAPK) in isolated rat pancreatic acini. The present study evaluates whether one or both types of CCK receptors are capable of MAPK activation in pancreatic AR42J acinar cells as well as CHO cells transfected with CCK-A or CCK-B receptors. CCK significantly increased p44 MAPK and p42 MAPK activities in AR42J cells. Minimal, half-maximal, and maximal responses were observed at 30 and 500 pM and 10 nM, respectively, after CCK-8 stimulation and at 100 pM and 1.5 and 30 nM, respectively, after gastrin stimulation. Glycine-extended gastrin had no effect at 100 nM and a small but significant effect at 1 microM. The CCK-B receptor antagonist L365,260 almost totally blocked MAPK activation in AR42J cells after stimulation with gastrin and glycine-extended gastrin and substantially reduced the activation of both kinases by CCK-8, while the CCK-A receptor antagonist L364,718 was much less effective. The CCK-A-selective agonist A71376, however, was an effective stimulant of MAPK activity. In an alternative approach, stably transfected CHO cells bearing either CCK-A or CCK-B receptors were stimulated with CCK-8. Each receptor induced a time-dependent increase in activity of both MAPKs by five- to sixfold in CCK-A- and CCK-B-bearing cells. In conclusion, both CCK-A and CCK-B receptors activate MAPK in AR42J cells and in transfected CHO cells.

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCKA receptor. 125I-CCK binding to intact cells at 37 degrees C revealed two affinity states for CCK of Kd values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[gamma S], ATP and ATP[gamma S] but not AMP-PCP. ATP and ATP[gamma S] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states.

To relate specific effects on growth and transformation to activation of specific affinity states of the CCKA receptor stably expressed in CHO cells we compared responses to the CCK analogues JMV-180 and CCK8. CCK8 led to an inhibition of both cell proliferation and transformation. Effects on proliferation were indicated by a reduction of DNA synthesis and cell numbers. Effects on transformation were indicated by a reduction of colony formation in soft-agar. JMV-180 did not inhibit cell proliferation although a small inhibitory effect on DNA synthesis was observed. JMV-180 inhibited the maximal effects of CCK8 on cell proliferation and DNA synthesis. In contrast, JMV-180 substantially inhibited cell colony formation in soft-agar and did not inhibit the effects of CCK8 on this parameter. Collectively these data with receptor affinity state specific analogues indicated that inhibition of cell proliferation and growth in soft-agar can be attributed to activation of distinct affinity states. Thus, different second messengers are likely responsible for the inhibitory effects on anchorage-dependent and -independent growth.

Cholecystokinin (CCK) stimulates the growth of pancreatic acinar cells. However, the molecular mechanisms involved in this trophic action are unknown. CCK binds to both high- and low-affinity receptor states, and these two states appear to activate separate sets of intracellular messengers and have opposite effects on amylase release. JMV-180 is a CCK analogue that interacts in the rat with the high-affinity state as an agonist and the low-affinity state as an antagonist. In the current study, CCK octapeptide (CCK-8) and JMV-180 were tested for their ability to stimulate the growth of rat pancreatic acinar cells in primary culture. CCK-8 stimulated [3H]thymidine incorporation into DNA in a dose-dependent manner. Effects were observed with 0.3 nM, and maximal increases were seen at 3 nM CCK-8 (442 +/- 53% of control, n = 5, P < 0.01). JMV-180 also stimulated DNA synthesis. Effects were noted with 10 nM, and a maximal increase of 267 +/- 82% (n = 4, P < 0.01) of control was stimulated by 100 nM JMV-180. These data with JMV-180 indicate that the high-affinity receptor state for CCK is capable of stimulating DNA synthesis. However, within the same experiment the effects of CCK were always significantly greater than those of JMV-180. To test whether CCK has an additional effect through interactions with the low-affinity state, the effects of a combination of JMV-180 with a maximal dose of CCK-8 were examined. JMV-180 inhibited the maximal effect of CCK-8 in a dose-dependent manner with a maximal inhibition occurring with 1 microM JMV-180. The effects of the combination of 3 nM CCK-8 and 1 microM JMV-180 were no greater than those of JMV-180 alone. Taken together these data indicate that CCK-mediated increases in DNA synthesis in rat pancreatic acinar cells in vitro occur by interactions with both high- and low-affinity receptor states.

In order to investigate the molecular characteristics of gastrointestinal hormone receptors, we have expressed their mRNAs in Xenopus laevis oocytes. Xenopus oocytes possess intrinsic muscarinic cholinergic receptors which couple to intracellular Ca2+ release. Release of intracellular Ca2+ was detected by an increase in 45Ca2+ release from preloaded oocytes or by using the Ca2+-sensitive fluorescent dye fura-2. Similarly, cholecystokinin (CCK) receptors expressed on the surface of oocytes which were injected with mRNA prepared from rat pancreatic acinar AR42J cells were readily detected by their ability to mobilize intracellular Ca2+. The CCK receptors expressed in oocytes showed similar binding characteristics as the native receptors. CCK receptor expression in the oocytes could be specifically blocked by hybridizing the mRNA with antisense oligonucleotides based on the highly conserved second transmembrane region of the HM4 muscarinic cholinergic receptor before injection. These latter results strongly suggest that the CCK receptor is a member of the G protein-linked receptor family. Thus, expression in Xenopus laevis oocytes provides a powerful tool for elucidation of the molecular characteristics of gastrointestinal hormone receptors.

The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2+-mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of 45Ca2+. CCK receptors were present 1 day (maximum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte CCK receptors retained selectivity for CCK analogs (CCK8 greater than unsulfated CCK8 greater than CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was subjected to size fractionation on sucrose gradients, activity-inducing CCK receptors showed a single peak centered at 3 kilobases. The generality of this oocyte system for expressing Ca2+-mobilizing hormone receptors was further shown by expression of a response to bombesin after injection of AR42J cell RNA and a response to vasopressin and angiotensin II when poly(A)+ RNA from rat liver was injected. No response to CCK was demonstrable after injection of liver RNA, demonstrating the specificity of this assay.