New insights into neurohormonal regulation of pancreatic secretion

GASTROENTEROLOGY 2004;127:957–969

SPECIAL REPORTS AND REVIEWS New Insights Into Neurohormonal Regulation of Pancreatic Secretion CHUNG OWYANG* and CRAIG D. LOGSDON‡ *Division of Gastroenterology, Department of Internal Medicine, and ‡Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

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.

he mediation of postprandial pancreatic enzyme secretion has been ascribed mainly to the peptides cholecystokinin (CCK) and serotonin (5-hydroxytryptamine; 5-HT) and to the vago-vagal reflex, which activates cholinergic postganglionic neurons in the pancreas.

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Knowledge of these regulatory mechanisms is considerable; however, evidence that both CCK and 5-HT act via the vagal cholinergic pathways to mediate pancreatic enzyme secretion suggests a more complex picture. Furthermore, vagal afferent signals evoked by CCK appear to be enhanced by 5-HT to stimulate pancreatic secretion. This review will discuss the current understanding of neurohormonal control of pancreatic enzyme secretion. CCK actions, both direct and neural, have been shown to mediate pancreatic secretion in rats, whereas, in humans, CCK appears to act entirely via the vagal cholinergic pathway. We will compare in vitro and in vivo actions of CCK on pancreatic secretion in rodents and humans. Conclusive evidence that human pancreatic acini lack functional CCK-A receptors will be presented, as will structural and functional evidence that CCK acts via the vagal afferent pathway to mediate pancreatic enzyme secretion. 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. The mechanisms by which nonCCK-dependent duodenal stimuli evoke pancreatic enzyme secretion acting through intestinal 5-HT will also be reviewed. The synergistic interaction between CCK and 5-HT in the mediation of postprandial pancreatic secretion will be discussed. Important physiologic ramifications notwithstanding, these findings explain the discrepancies observed in previous in vivo vs. in vitro studies, and they revolutionize our current concept of the mechanism of action of CCK on pancreatic exocrine secretion. Abbreviations used in this paper: CCK, cholecystokinin; CCK-8, cholecystokinin octapeptide; 5-HT, 5-hydroxytryptamine, serotonin. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gastro.2004.05.002

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In Vitro Action of CCK on Pancreatic Acini CCK acts directly on pancreatic acini to stimulate pancreatic secretion or so researchers have believed. Most data supporting this belief were obtained using dispersed pancreatic acini from rodents. CCK-A receptors are known to preside on pancreatic acini and exist in both high- and low-affinity states.1,2 A third CCK-A receptor, also existing in a low-affinity state, has been proposed.3 In vitro studies have shown that CCK-A receptors are highly sensitive to CCK at levels as low as 1 pmol/L. Binding of CCK to CCK-A receptors on rat pancreatic acinar cells leads to an increase in the concentration of intracellular Ca2⫹ and an increase in digestive enzyme secretion in vitro.2 These observations, together with the knowledge that atropine does not alter the enzyme response to CCK in isolated pancreatic acini4 and that the effects of cholinergic agonists and CCK in such preparations are merely additive,4,5 suggest that the effect of CCK on pancreatic enzyme secretion is not cholinergically dependent. Hence, in rodents, at least 1 mechanism by which CCK stimulates pancreatic enzyme secretion is by direct action on CCK-A receptors that are expressed on pancreatic acinar cells. Whether this direct mechanism functions in humans has been difficult to ascertain because of the controversy regarding the presence and identity of CCK receptors on human pancreatic acinar cells.

Human Pancreatic Acini Lack Functional CCK-A Receptors Receptor presence can be established biochemically, at the level of receptor mRNA or protein, or functionally. The expression of CCK-A receptors on rodent pancreatic acinar cells was readily established by each of these criteria. Multiple approaches, including radioligand binding to isolated acini,6,7 receptor crosslinking,8 receptor autoradiography in situ,9 Northern blot analysis,10 –12 polymerase chain reaction studies,11 and in vitro secretion studies4 identified the CCK receptor on rat pancreatic acinar cells to be the CCK-A subtype. In contrast, the presence, let alone the type, of CCK receptors in the human pancreas has been difficult to verify using any criterion. Early attempts using Northern blot analysis10 and the sensitive method of reverse-transcriptase polymerase chain reaction13 failed to detect CCK-A receptor mRNA in the human pancreas. Researchers eventually succeeded in detecting CCK receptors using reverse-transcriptase polymerase chain reaction,11,14,15 but, unlike the rodent pancreas, CCK-B

Figure 1. Reverse-transcriptase polymerase chain reaction (RT-PCR) amplifies both CCK-A and CCK-B receptor mRNA from total RNA prepared from human whole pancreas and isolated acini. RT-PCR was performed using 70 ng DNase-purified RNA; the results are representative of 3 independent experiments. Sequencing verified that the bands amplified represented the expected genes. Note that standard RT-PCR indicates the presence of very low levels of message for the CCK-A receptors and much higher levels of CCK-B receptor, m3 muscarinic acetylcholine receptor, and gastrin-binding protein (GBP) mRNA. INS, insulin mRNA. Reprinted with permission from Ji et al.14

rather than CCK-A receptor mRNA was found to be predominant (Figure 1). In this regard, humans resemble several other species, including calves,16 pigs,17 and dogs.18,19 Receptor autoradiography confirmed this finding; CCK-B receptors but not CCK-A receptors were detected in sections of human pancreas.20 Quantitative polymerase chain reaction methods have since explained the difficulties encountered in detecting CCK-A receptor messenger RNA (mRNA) in human samples. Low levels of mRNA for both CCK receptor subtypes were measured in human pancreas.21 CCK-B receptor mRNA levels were at least 10-fold lower than mRNA levels for m3 muscarinic cholinergic receptors in human pancreas. In rats, by comparison, CCK-A receptor RNA levels were similar to m3 muscarinic cholinergic receptor mRNA levels. Furthermore, in human samples, mRNA levels for CCK-A receptors were much lower than those for CCK-B receptors and in fact were estimated to be less than 1 copy per acinar cell. Therefore, studies of human acinar cells at the level of mRNA did not support the expression of CCK-A receptors in any significant amount but suggested rather the presence, albeit at low levels, of CCK-B receptors. The expression of CCK-B receptors on human pancreatic acinar cells has been difficult to corroborate. Immunohistochemical studies failed to detect significant CCK-B expression on human pancreatic acinar cells; those receptors observed appeared to be localized to cells within the pancreatic islets of Langerhans.22–24 Specifically, CCK-B receptors were reported to exist on the somatostatin-secreting D cells,23,24 and CCK-A receptors were observed on insulin-secreting ␤ cells in fetal human pancreas.22 Conclusive evidence for the presence of either CCK-A or CCK-B receptors on human acinar cells could not be found.

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Figure 2. Human pancreatic acini secrete amylase in response to carbachol but not CCK-8 or gastrin. Isolated human pancreatic acini, either uninfected or infected for 4 hours with an adenovirus that expresses the human CCK-B receptor (AdCCKB), were incubated with increasing concentrations of CCK-8, gastrin, or carbachol at 37°C for 30 minutes. The concentration of amylase released into the medium was measured using a colorimetric reagent and was expressed as a percentage of initial acinar amylase content. Data are the means of 3 separate experiments. Reprinted with permission from Ji et al.14

The most compelling evidence that human pancreatic acinar cells lack CCK receptors has come from functional studies. Early attempts to isolate human acini resulted in preparations that showed low levels of response to stimulation; the isolated acini appeared to respond only to pharmacologically high concentrations of either CCK or the acetylcholine agonist carbachol.25 The difficulties of obtaining viable samples of normal human pancreas combined with the formidable challenge of isolating functional preparations of acini were thought to explain this apparent lack of responsiveness. Subsequently, preparations of normal human acini were produced that showed robust secretory responses to activation of muscarinic cholinergic receptors and no response to stimulation with CCK.14,26 Additional evidence of the functionality of the isolated human acinar cells came from experiments indicating that adenoviral-mediated transfer of CCK-A receptor cDNA resulted in full secretory responses to CCK stimulation (Figure 2).14 Furthermore, an increase in the concentration of intracellular Ca2⫹ was measured in isolated human acini treated with CCK, after, but not before, CCK receptor gene transfer (Figure 3).14 Because changes in intracellular Ca2⫹ are a sensitive biologic indicator of CCK receptor activation, these data strongly indicate the lack of direct functional responses to CCK in human pancreatic acinar cells.

Role of Cholinergic Pathways in Pancreatic Enzyme Secretion Experimental evidence suggests that cholinergic pathways play a major role in mediating pancreatic se-

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cretion under physiologic conditions. Konturek et al.27 demonstrated that blockade of the cholinergic nervous system suppressed basal pancreatic secretion in dogs. Furthermore, atropine inhibited 80% of the pancreatic protein output evoked by intraduodenal perfusion of an amino acid mixture. Others confirmed inhibition of amino acid- and oleate-evoked enzyme output.28 –31 Numerous investigators, however, failed to demonstrate atropine inhibition of CCK- or CCK-like peptide-evoked pancreatic enzyme secretion in the dog.29,32 None of these studies assessed whether the high doses of CCK typically used reproduced physiologic conditions. Konturek et al.27 reported that atropine inhibited pancreatic enzyme secretion induced by low doses of exogenous CCK in dogs, whereas enzyme output in response to high doses of CCK was relatively insensitive to atropine. Soudah et al.33 and others34,35 reported similar observations in humans; in healthy subjects, CCK infusion that produces plasma CCK levels similar to those seen postprandially stimulates pancreatic secretion by an atropinesensitive pathway (Figure 4). Considered together, these observations suggest that, in experimental animals and in humans, cholinergic neural pathways rather than pancreatic acini represent the primary targets in which CCK acts to stimulate pancreatic enzyme secretion.

CCK Acts Via the Vagal Afferent Pathways to Mediate Pancreatic Secretion Atropine and hexamethonium completely abolish pancreatic enzyme responses to physiologic doses of CCK in rats, which suggests that CCK acts on a presynaptic

Figure 3. Intracellular Ca2⫹ levels increase in human pancreatic acini in response to carbachol but not to CCK-8. An estimate of intracellular Ca2⫹ was obtained by measuring emitted fluorescence from fura-2loaded acini using an Attofluor digital imaging system. CCK-8 (100 nmol/L) or carbachol (1 mmol/L) was introduced at the times indicated. (A) Freshly prepared acini were treated with CCK-8 and then carbachol. Acini infected for 4 hours with an adenovirus encoded with either human CCK-B receptor (B) or rat CCK-A receptor (C) were treated with CCK-8 (100 nmol/L). Data shown are typical and are representative of 4 separate experiments. Reprinted with permission from Ji et al.14

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Figure 4. In humans, CCK-8 infusion evokes a dose-dependent increase in net output of both trypsin (A) and lipase (B). Atropine administration nearly abolishes CCK-8 –stimulated enzyme release at CCK-8 doses of 5 and 10 ng·kg⫺1·h⫺1 but is relatively less potent at higher doses, i.e., 20 and 40 ng·kg⫺1·h⫺1 (f ⬍ 0.01, analysis of variance for repeated measures; results are means ⫾ SE, n ⫽ 6). Reprinted with permission from Soudah et al.33

site along the cholinergic pathway.36 Vagotomy also completely abolishes pancreatic responses to CCK stimulation but has little effect on the pancreatic response to supraphysiologic doses of CCK.36 The proposal that direct stimulation of pancreatic acinar secretion by CCK requires a basal vagal cholinergic tone was ruled out by the demonstration that, after vagotomy, CCK did not evoke an additional increase in pancreatic secretion stimulated by bethanechol.36 This result suggests rather that CCK stimulates pancreatic secretion via vagal pathways, a premise supported by previous reports that truncal vagotomy leads to reduced basal and nutrient-stimulated pancreatic secretion.34,37 These findings must be contrasted with earlier reports that vagotomy did not significantly reduce pancreatic enzyme secretion in response

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to exogenous CCK.38 – 40 Contradictory findings such as these may be the result of different lengths of time postvagotomy prior to taking experimental measurements and, in the earlier studies, the use of supraphysiologic doses of CCK. The pancreas quickly regains function when challenged with vagal injury.41 This extraordinary plasticity may explain both the profound effect36,37 and the absence of effect of vagotomy on pancreatic secretion.38,40,42 After chronic vagotomy, pancreatic protein secretion in response to CCK-8 stimulation is fully restored by day 20 in both anesthetized and conscious rat models.43,44 In contrast to its effect in vagally intact rats, atropine fails to inhibit pancreatic secretion stimulated by CCK in chronically vagotomized rats.43,44 On the other hand, hexamethonium treatment and surgical interruption of the enteropancreatic neural connection each markedly reduce pancreatic responses to CCK. Moreover, application of benzalkonium chloride to the duodenal serosa ablates myenteric neurons, as one would expect, and also abolishes CCK-8-stimulated pancreatic secretion. A potent gastrin-releasing peptide receptor antagonist was shown to reduce markedly the pancreatic response to CCK in chronically vagotomized rats but to have no effect in vagally intact rats.43 Immunohistochemical studies have demonstrated that CCK-8 administration evokes an increase in the percentage of c-Fos–positive pancreatic neurons containing choline acetyltransferase in vagally intact rats, whereas, after chronic vagotomy, the number of c-Fos-positive neurons containing gastrinreleasing peptide increases. These observations support the hypothesis that neural remodeling occurs after chronic vagotomy; intraduodenal cholinergic neurons are recruited that become responsive to CCK-8 and activate an intrapancreatic gastrin-releasing peptide neural pathway to mediate pancreatic secretion (Figure 5).43 Again, the variable effects of vagotomy on pancreatic secretion likely are related to the different lengths of time between vagotomy and the recording of experimental measurements. CCK acts via vagal afferent pathways to induce satiety45 and decrease gastric emptying.46 Similar mechanisms apply to pancreatic enzyme secretion. Perivagal pretreatment with capsaicin, a sensory neurotoxin that targets small-diameter sensory neurons,47 impairs pancreatic responses to physiologic doses of CCK, an effect similar to that observed with vagotomy or administration of atropine (Figure 6).36 Further studies have shown that gastroduodenal, but not jejunal, application of capsaicin abolishes pancreatic secretion in response to physiologic doses of CCK.36 This indicates that CCK stimulates pancreatic enzyme secretion via a capsaicin-sensitive

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Figure 5. (A) Sites and mechanisms of action of CCK to stimulate pancreatic secretion in rats. Physiologic levels of CCK in plasma act via stimulation of the vagal afferent pathways. In contrast, supraphysiologic plasma CCK levels act on intrapancreatic neurons and to a larger extent on pancreatic acini. (B) Adaptive changes that occur after chronic vagotomy involve recruitment of intraduodenal cholinergic neurons that activate a gastrin-releasing peptide neural pathway to stimulate secretion. ACh, acetylcholine. Reprinted with permission from Li and Owyang.41

afferent vagal pathway that originates in the gastroduodenal mucosa (Figure 5). Endogenously released CCK is heterogenous and consists of multiple molecular forms such as CCK-58, CCK33, CCK-8, and other intermediate forms.48 It is conceivable that different forms of CCK have a preferential site of action when mediating pancreatic enzyme secretion. In fact, blood from fasted rats and rats in which CCK released was stimulated by the trypsin inhibitor camostat contained mainly CCK-58.49 The mechanism by which CCK-58 stimulates pancreatic enzyme secretion in rats has not been clearly defined. However, using 2 different experimental rat models that evoke endogenous CCK release (diversion of bile-pancreatic juice50 –52 and intraduodenal infusion of casein),53,54 it has been shown that both perivagal application of capsaicin and vagal afferent rootlet section abolish the increase in pancreatic secretion.51 This indicates that endogenous CCK, including CCK-58, similar to exogenous CCK, stimulates the vagal afferent pathway to mediate pancreatic exocrine secretion. Recently, Yamamoto et al.55 reported that, similar to the results of Li and Owyang,36,52 chemical ablation of vagal afferent fibers with capsaicin abolished the effect of exogenous and endogenous CCK on pancreatic secretion. However, perivagal application of capsaicin showed no influence on pancreatic growth stimulated by CCK.55 These findings suggest that exogenous and endogenous CCK stimulate pancreatic growth not via capsaicin-sensitive vagal afferent pathways but through capsaicininsensitive vagal fibers or directly on the pancreas in rats.

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The findings of Li and Owyang36,52 and Yamatoto et al.55 should be contrasted with studies from 2 other laboratories. In conscious rats, Masuda et al.56 and Guan et al.57 showed that CCK’s action on pancreatic secretion was not affected by perivagal application of capsaicin. Similarly, 2 preliminary studies by Coskun et al.58,59 also failed to confirm that pancreatic secretion stimulated by CCK is mediated by capsaicin-sensitive vagal pathways in anesthetized rats. The disparate findings from different laboratories may be explained by the recent report that CCK may activate both A (capsaicin resistant) and C (capsaicin sensitive) type vagal afferent neurons.60 Guan et al.57 used 10-fold less capsaicin to induce neuronal degeneration than Li et al.36,52 The study by Guan et al.57 demonstrated that their capsaicin treatment was sufficient to prevent inhibition of food intake and gastric emptying by CCK. It is conceivable that, in vagal afferent neurons, there may be a range of sensitivities to destruction by capsaicin treatment. CCK-induced pancreatic secretion may rely on vagal sensory neurons that are more resistant to capsaicin than those mediating inhibition of food intake and gastric emptying. In addition, the physiologic control of pancreatic secretion in anesthetized and conscious rats may differ. Previous studies have reported that, in contrast to responses elicited by atropine in the anesthetized rat, which appear to be similar to the human responses, atropine does not significantly affect the net increase in pancreatic secretion evoked by physiologic doses of CCK in the conscious rat.39,61 A complex interaction between neural and humoral stimulatory influences likely leads to the high level of basal pancreatic secretion in the conscious rat. Atropine markedly inhibits basal pancreatic secretion, which

Figure 6. In rats, perivagal application of capsaicin and vagal afferent rootlet section each completely abolish the pancreatic response to physiological doses of CCK-8 (i.e., ⱕ40 pmol·kg⫺1·h⫺1), but neither affects the pancreatic response to supraphysiologic doses of CCK. Values are mean ⫾ SE, n ⫽ 6 in each group. *P ⬍ 0.05. Reprinted with permission from Li and Owyang.36

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results in a reduction of the total pancreatic output evoked by CCK, thus affecting the net increase.39 Atropine is likely to produce widespread cholinergic blockage in the central and enteric nervous systems, which alters normal gastrointestinal physiology. For example, atropine enhances the pancreatic response to a protein meal that was medicated by CCK.38 The increase in CCK secretion secondary to suppression of basal pancreatic secretion by atropine may, in turn, stimulate pancreatic enzyme secretion. The atropine experiments in conscious rats therefore do not necessarily negate the importance of vagal mediation in the action of CCK in that generalized suppression of cholinergic tone may mask the action of CCK on the vagal afferent pathway. In fact, in a recent in vivo study of conscious rats, perivagal capsaicin treatment completely abolished the pancreatic response to CCK stimulation.62

Structural and Functional Evidence That CCK Acts on Vagal Afferent Fibers CCK receptors have been detected in the rat vagus nerve using in vitro receptor autoradiography.63 Nerve ligation experiments have shown that these receptors are transported toward the peripheral nerve endings from the nodose ganglion.63 CCK binding and axonal transport are evident in all abdominal vagal branches.64 The CCK receptors are predominantly type A64 because the CCK-A receptor antagonist L-364,718 completely abolishes 125I-CCK binding, and nonsulfated CCK has no effect. CCK-A receptors are also present in the nucleus of the solitary tract,65 which is the terminal for the central projections of vagal afferents from the nodose ganglion. Unilateral nodosectomy or unilateral transection of afferent vagal rootlets causes a marked unilateral reduction in CCK binding in the nucleus of the solitary tract,66 which indicates that CCK receptors are localized presynaptically on vagal afferent terminals. These observations are consistent with the notion that CCK receptors preside on the vagal afferents. Perivagal capsaicin treatment markedly reduces the binding of CCK on the vagal nerve, providing further confirmation.67 Anterograde transport of CCK receptors appears to be bidirectional, from the afferent cell bodies in the nodose ganglion to vagal afferent terminals in the nucleus of the solitary tract and toward the peripheral vagal afferent fibers, which may serve as target sites for the mediation of CCK action on pancreatic secretion. Electrophysiologic studies in rats and ferrets have provided evidence that CCK stimulates vagal afferent

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pathways.67,68 Li et al.67 recorded the unitary activities of sensory vagal neurons using microelectrodes implanted in rat nodose ganglia. CCK infusion at 40 pmol kg⫺1 hr⫺1, which mimicks postprandial levels, evoked a marked increase in discharge over basal.67 A short latency, slow adaptation, and rapid return to basal on removal of the stimulus characterized the response. Atropine, which had no affect on this response, abolished all gastroduodenal motor activities, indicating that the response to CCK is not caused by gastrointestinal contractions. Gastrointestinal contractions are abolished by vagotomy, perivagal capsaicin treatment, and application of capsaicin to the gastroduodenal mucosa, but they are not affected by supranodose vagotomy. Similar studies in ferrets68 showed that mucosal vagal afferent fibers from the duodenum are highly sensitive to CCK-8. Evocative luminal stimuli included light stroking and hypertonicity. Electrophysiologic studies together with receptor autoradiography studies provide functional and structural evidence that CCK acts on vagal afferent pathways.

CCK-A Receptor Affinity States and Ca2ⴙ Signal Transduction in Vagal Nodose Ganglia In rat pancreatic acini, CCK has been shown to interact with 2 affinity states of the CCK-A receptor.1,69 One site of interaction, characterized by high affinity and low capacity, is associated with stimulation of enzyme release at CCK concentrations up to 100 pmol/L. The other, a low-affinity and high-capacity site, is thought to mediate the inhibition of CCK-mediated enzyme release. It is not known whether these 2 sites represent distinct proteins or different affinity states of the same receptor protein. Galas et al.70 developed a series of CCK analogs (e.g., CCK-JMV-180) in which substitutions of the Asp32-Phe33 region produced molecules lacking the primary amide function. These compounds exhibit a unique pharmacologic profile in rats, both in vitro and in vivo: high-affinity CCK-A receptor agonists, which stimulate amylase release; and low-affinity functional antagonists, which block CCK-induced supramaximal inhibition of amylase release.71 Thus, CCK-JMV-180 can be used to distinguish actions mediated by high-affinity CCK receptors from those mediated by low-affinity CCK receptors. Electrophysiologic evidence for high- and low-affinity vagal CCK-A receptors has come from studies that involve the recording of single-unit discharges of sensory neurons from the nodose ganglia that supply the gastrointestinal tract.72 The CCK analog CCK-JMV-180, which acts as an agonist on high-affinity CCK receptors

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These results suggest the presence of high- and lowaffinity states of CCK-A receptors on nodose ganglia.73 The Ca2⫹-signaling modes appear to be mediated through L-type Ca2⫹ channels and to involve the participation of Gq proteins.73

High-Affinity CCK-A Receptors on the Vagus Nerve Mediate CCKStimulated Pancreatic Enzyme Secretion

Figure 7. Effect of CCK-JMV-180 on the response of neurons from rat nodose ganglia to the diversion of bile-pancreatic juice (BPJ). Eight neurons that responded to a 60-minute BPJ diversion were tested. Administration of CCK-JMV-180 abolished the response to BPJ diversion in 3 of 8 neurons but had no effect on the response in the other 5 neurons. These observations suggest that endogenous CCK acts on both high- and low-affinity CCK-A receptors. *P ⬍ 0.05. Reprinted with permission from Li et al.72

and as an antagonist on low-affinity CCK receptors, was used to identify the vagal CCK receptor affinity states involved in the mediation of the vagal afferent response to endogenously released CCK evoked by the diversion of bile-pancreatic juice in rats.72 Seven of 32 single units were stimulated by the bile-pancreatic juice diversion. The responses were abolished by acute subdiaphragmatic vagotomy or perivagal capsaicin treatment. The CCK-A receptor antagonist CR-1409, but not the CCK-B antagonist L-365,260, blocked the vagal response to endogenous CCK stimulation. Infusion of CCK-JMV-180 completely blocked the vagal afferent response to the diversion of bile-pancreatic juice in 3 of 8 neurons tested and had no effect on the response in the remaining 5 (Figure 7). Gastric, celiac, and hepatic branch vagotomy each abolished the response in different subgroups of neurons. These studies confirm the presence of both high- and low-affinity CCK-A receptors on distinct vagal afferent fibers. To characterize the mode of Ca2⫹ signal transduction activated by CCK, in vitro Ca2⫹ signaling studies were performed using acutely isolated rat nodose ganglion cells.73 CCK-8 at a concentration of 1 nmol/L primarily evoked a Ca2⫹ transient, which was followed by a sustained Ca2⫹ plateau (45% of cells responded), whereas 10 pmol/L CCK-8 evoked Ca2⫹ oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and lowaffinity antagonist of CCK-A receptors, primarily elicited Ca2⫹ oscillations. CCK-OPE inhibited the Ca2⫹ transient induced by 1 nmol/L CCK-8 but not that induced by carbachol and a high concentration of K⫹.

CCK-A receptors are present on all abdominal vagal branches.64 These receptors may serve as target sites for the mediation of CCK actions. There is evidence that CCK stimulates vagal afferent fiber discharge, which alters proximal gastric motility and emptying.46 Similarly, afferent fibers of the abdominal vagus nerve mediate CCK-elicited satiety signals to the area postrema and the nucleus of the solitary tract of the dorsal hindbrain.74,75 Because vagal CCK receptors exist in both high- and low-affinity states, it is conceivable that different affinity states of the vagal CCK receptors mediate different digestive functions. To identify the vagal CCK receptor affinity state involved in the mediation of pancreatic secretion, in vivo rat studies using CCK-JMV-180 have been performed.76 The CCK-A receptor antagonist L-364,718 blocked dose-dependent increases in pancreatic secretion evoked by CCK-JMV-180. Acute vagotomy in anesthetized rats and perivagal application of capsaicin in conscious rats abolished this response, indicating that CCK-JMV-180 stimulates pancreatic secretion by the vagal afferent pathway. CCK-JMV-180 failed to block the pancreatic response to physiologic doses of CCK-8 (Figure 8). Furthermore, in conscious rats, CCK-JMV-180 enhanced rather than inhibited pancreatic protein secretion in re-

Figure 8. Effects of CCK-JMV-180 on CCK-8 –induced pancreatic secretion in anesthetized rats. (A) Intravenous infusion of CCK-8 at 20 and 40 pmol·kg⫺1·h⫺1 increases protein secretion by 50% and 78% over basal, respectively. CCK-JMV-180 alone, 22 and 44 ␮g·kg⫺1·h⫺1, stimulates pancreatic protein secretion by 48% and 82% over basal, respectively. (B) CCK-JMV-180 does not inhibit but significantly enhances the pancreatic response to CCK-8. Values are means ⫾ SE, n ⫽ 6 rats in each group. *P ⬍ 0.05 compared with infusion of CCK-8 alone. Reprinted with permission from Li et al.76

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Figure 9. Pancreatic protein secretion induced by administration of casein and CCK-JMV-180. Duodenal infusion of 18% casein in conscious rats produces a 2.5-fold increase in protein secretion. Perivagal application of capsaicin abolishes the pancreatic response to casein. CCK-JMV-180 infusion (44 ␮g·kg⫺1·h⫺1) fails to inhibit the pancreatic response to intraduodenal casein infusion, which suggests that endogenous CCK acts on high- but not low-affinity CCK-A receptors to stimulate pancreatic secretion. *P ⬍ 0.05. Reprinted with permission from Li et al.76

sponse to intraduodenal administration of 18% casein, which had been shown to release endogenous CCK (Figure 9).76 These observations indicate that both exogenous and endogenous CCK evoke pancreatic secretion by acting on high-affinity, not low-affinity, CCK receptors. The action of CCK on pancreatic secretion must be contrasted with its effects on gastric mechanosensitive vagal afferent fibers77 and on satiety.78 Weatherford et al.78 demonstrated that a CCK-JMV-180 dose dependently reverses the effect of CCK-8 on satiety. Maximal effects were obtained with a CCK-JMV-180 dose 1000 times higher than the CCK-8 dose. This suggests that the anorexic activity of CCK is mediated through interaction with a receptor site pharmacologically similar to the pancreatic low-affinity CCK receptor site. In another study, Schwartz et al.77 reported that CCK-JMV-180 completely blocked the gastric mechanosensitive vagal afferent response to arterial infusion of CCK-8, which suggests that low-affinity CCK receptors also mediate this response. Hence, vagal CCK receptors clearly exist in different affinity states and mediate different digestive functions.

CCK and 5-HT Interact Synergistically to Mediate Postprandial Pancreatic Secretion Postprandial pancreatic secretion is mainly controlled by the hormone CCK and intestinal 5-HT, which acts as a paracrine substance to stimulate the vago-vagal reflex that activates cholinergic postganglionic neurons

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in the pancreas.79 In the rat, administration of the CCK-A receptor antagonist L-364,718 inhibits postprandial pancreatic enzyme secretion by 54%, whereas the combination of L-364,718 and the 5-HT antagonists ICS-205,930, a 5-HT3 antagonist and ketanserin, a 5-HT2 antagonist, inhibits secretion by 94%. 5-HT thus appears to play a critical role in mediating postprandial pancreatic secretion. Electrophysiologic studies have clearly shown that 5-HT and ␣-methyl-5-HT activate 5-HT3 receptors located on the nerve terminals within the mucosa of ferret stomach and duodenum, which leads to the firing of vagal afferent fibers.80 – 82 Hillsley et al.83 reported that 5-HT activates different groups of afferent fibers innervating the rat jejunum: one group of mucosal nerve fibers is activated directly by stimulation of 5-HT3 receptors, and another group responds to contractile activities induced by stimulating 5-HT2A receptors on smooth muscle cells (i.e., mechanosensitive afferents). Using the nodose ganglia recording technique in anesthetized rats, Zhu et al.84 showed that luminal factors such as intestinal osmotic stimuli and perfusion of carbohydrates elicit powerful vagal nodose responses that are antagonized by the 5-HT3/4 antagonist tropisetron or the 5-HT3 antagonist granisetron (Figures 10 and 11). Similarly, intraluminal infusion of 5-HT elicits an increase in vagal afferent discharge by activating 5-HT3 receptors. Pharmacologic depletion of 5-HT stores using p-chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, abolishes nodose neuronal responses stimulated by luminal factors. In contrast, pretreatment with 5,7-dihydroxytryptamine, a specific neurotoxin that destroys 5-HT-containing neurons without affecting 5-HT-containing mucosal cells, has no effect on these responses. These findings suggest that the nodal neuronal responses to luminal osmolarity and the digestion products of carbohydrates depend on the release of endogenous 5-HT from the mucosal enterochromaffin cells, which acts on 5-HT3 receptors on vagal afferent fibers. Luminal factors such as osmolarity and disaccharides stimulate pancreatic secretion by activating 5-HT3 receptors, whereas mechanical stimulation activates both 5-HT3 and 5-HT2 receptors on mucosal vagal afferent fibers in the intestine to stimulate pancreatic secretion.79 Luminal perfusion of maltose or hypertonic NaCl in vivo leads to a 3-fold increase in the 5-HT level in rat intestinal effluent perfusates.85 Intraluminal perfusion of 5-HT (10⫺5 mol/L) evokes a similar increase. Intraduodenal administration of 5-HT evokes a dose-dependent increase in pancreatic protein secretion, which is not blocked by the CCK-A antagonist CR-1409. Acute vagotomy, administration of methscopolamine, or periva-

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Figure 10. Action potentials of a rat nodose ganglia neuron that innervates the duodenal mucosa, after intraduodenal infusion of hypertonic NaCl. (A) Control: nodose neurons that innervate the duodenal mucosa have a very low basal level of activity. Duodenal infusion of normal saline does not affect vagal afferent discharge. (B) Duodenal infusion of hypertonic NaCl (500 mOsm/L, 2 mL/min) produces a marked increase in vagal afferent discharge, which peaks at 26 impulses within 20 seconds and returns to basal 20 seconds after the lumen is rinsed with isotonic saline. (C) Administration of the 5-HT3 receptor antagonist granisetron abolishes the nodose neuronal response evoked by hypertonic NaCl. Each action potential is portrayed as a standard pulse (upper line) and an original tracing (lower line). Reprinted with permission from Zhu et al.84

gal or intestinal application of capsaicin abolishes 5-HTinduced pancreatic secretion.85 In conscious rats, intraluminal administration of 5-HT (10⫺5 mol/L) produces a 90% increase in pancreatic protein output, which is markedly inhibited by the 5-HT3 antagonist ondansetron. These observations indicate that luminal stimuli induce 5-HT release, which in turn activates 5-HT3 receptors on mucosal vagal afferent terminals. In this manner, 5-HT acts as a paracrine substance to stimulate pancreatic secretion via a vagal cholinergic pathway. Thus, it appears that both 5-HT and CCK evoke pancreatic enzyme secretion by stimulating vagal afferent pathways that originate in the duodenal mucosa. Electrophysiologic studies in the ferret substantiate this observation. The ferret small intestine is endowed with a rich supply of mucosal afferent fibers that are sensitive to luminal stimuli such as hypertonic saline, HCl, and light stroking,82 which may stimulate the vagal afferents by acting through 5-HT. Some of these fibers also are sensitive to CCK. Thus, CCK and 5-HT may act on the same or distinct vagal afferent fibers in the mucosa. This interaction between CCK and nonCCK-dependent stimuli on pancreatic secretion was in-

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vestigated using an in vivo rat model.62 Infusion of a subthreshold dose of CCK-8 (15 pmol kg⫺1 hr⫺1) by itself failed to stimulate pancreatic secretion. However, the same infusion accompanied by an intraduodenal perfusion of maltose or hypertonic saline evoked significantly higher levels of pancreatic secretion than observed with either maltose or hypertonic saline alone (Figure 12).62 This suggests that CCK potentiates non-CCKdependent stimuli, which act through 5-HT to stimulate pancreatic secretion. To characterize further the interaction between CCK and 5-HT, single neuronal discharges of rat vagal primary afferent neurons innervating the duodenum were recorded.86 Two groups of nodose ganglia neurons emerged: group A neurons, which respond to intraarterial injections of low doses of CCK-8 (i.e., 10 – 60 pmol/L); and group B neurons, which in addition to responding only to high doses of CCK-8 (120 –240 pmol/L), are also activated by duodenal distention. CCKJMV-180 stimulates group A nodose neurons in a dosedependent manner, but inhibits the effects of CCK-8 at high doses on group B neurons, which indicates that CCK-A receptors exist in high-and low-affinity states on distinct nodose neurons. In the same study, it was ob-

Figure 11. Response of a rat nodose ganglia neuron to intraduodenal infusion of maltose. (A) The neuron responds to maltose after a latency of 10 seconds. Activity lasts for the duration of the stimulus and ceases when the lumen is rinsed with isotonic saline. (B) Administration of the CCK-8 antagonist CR-1409 has no effect on the nodose neuronal response to maltose. (C) Administration of the 5-HT3 receptor antagonist granisetron abolishes the nodose neuronal response evoked by maltose. Each action potential is portrayed as a standard pulse (upper line) and an original tracing (lower line). Reprinted with permission from Zhu et al.84

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that a very small increase in plasma CCK levels is sufficient to evoke a robust postprandial pancreatic secretion.

Conclusion

Figure 12. Interaction between CCK and non-CCK– dependent pancreatic stimuli in rats. A subthreshold dose of CCK (15 pmol kg⫺1 h⫺1) potentiates the pancreatic responses to intraduodenal administration of maltose (300 mmol/L) (A) and hypertonic NaCl (500 mOsm/L) (B). Values are means ⫾ SEM, n ⫽ 6 in each group. *P ⬍ 0.01. Reprinted with permission from Li and Owyang.62

served that intraduodenal perfusion of 5-HT produced a dose-dependent increase in nodose discharges. Some nodose neurons that responded to 5-HT failed to respond to either high or low doses of CCK-8. A separate group of nodose neurons possessing high-affinity CCK-A receptors also responded to luminal infusion of 5-HT. In this same group of neurons, a subthreshold dose of CCK-8 (5 pmol/L) produced no measureable electrophysiologic effects but enhanced the neuronal response to 5-HT. This potentiation effect of CCK-8 was eliminated by CR1409. Thus, the vagal nodose ganglia contain neurons that possess only high- or low-affinity CCK-A receptors or 5-HT3 receptors. However, there are neurons that express high-affinity CCK-A receptors and also 5-HT3 receptors. Prior exposure to luminal 5-HT may enhance the subsequent response to subthreshold doses of CCK. This synergistic interaction may explain the observation

Despite substantial differences between rodent and human pancreatic physiology, CCK appears to act via vagal cholinergic pathways to mediate pancreatic enzyme secretion in both species. Conclusive evidence that human pancreatic acini lack functional CCK-A receptors explains why a CCK infusion that produces plasma CCK levels similar to those seen postprandially stimulates pancreatic exocrine secretion by an atropinesensitive pathway. High- and low-affinity CCK receptors, although abundant on rat pancreatic acini, appear to play only a minor role in mediating pancreatic secretion. This seemingly incongruous relationship between in vitro observations and in vivo physiology is not uncommon. For instance, calcitonin gene-related peptide and somatostatin receptors are present on pancreatic acini but do not appear to mediate pancreatic secretion.87,88 These findings not only explain the discrepancies in previous in vivo vs. in vitro studies, but they revolutionize our current concept of the mechanism of action of CCK on pancreatic exocrine secretion. Under physiologic conditions, in both rodents and humans, cholinergic vagal afferent pathways rather than pancreatic acinar cells represent the primary targets on which CCK may act as a major mediator of postprandial pancreatic secretion. Although most of the information on vagal CCK-A receptors is obtained from research on rats, physiologic studies suggest that this information is probably applicable to humans. Experimental evidence indicates that, in contrast to the effect of CCK on satiety, which is mediated by low-affinity vagal CCK-A receptors, exogenous and endogenous CCK act through high-affinity vagal CCK-A receptors to mediate pancreatic secretion. The vagal afferent pathways also transmit sensory information about the mechanical and physiochemical state of the digestive tract, mediated in part by 5-HT, which in turn influences pancreatic secretion. A synergistic interaction between CCK and 5-HT at the level of the nodose ganglia may explain the robust postprandial pancreatic enzyme secretion despite a modest increase in plasma CCK after a meal. This supports the Pavlovian concept that the neural system is the major regulator of pancreatic secretion.

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Received December 2, 2003. Accepted April 29, 2004. Address requests for reprints to: Chung Owyang, M.D., Department of Internal Medicine, 3912 Taubman Center, University of Michigan Health System, Ann Arbor, Michigan 48109-0362. e-mail: [email protected]; fax: (734) 936-7392. Supported by National Institute of Diabetes and Digestive and Kidney Diseases grants RO1 DK48419 (to C.O.), DK32838 (to C.O.), and P30 34933 (to C.O.) and RO1 DK41225 (to C.D.L.).