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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2018 Jun 14;315(5):G734–G742. doi: 10.1152/ajpgi.00127.2018

Cholecystokinin induces esophageal longitudinal muscle contraction and transient lower esophageal sphincter relaxation in healthy humans

Arash Babaei 1,✉, Ravinder Mittal 2
PMCID: PMC6293254  PMID: 29902066

Abstract

Cholecystokinin (CCK) is known to cause lower esophageal sphincter (LES) relaxation through the activation of inhibitory motor neurons. CCK receptor agonists increase the frequency of transient LES relaxation through a peripheral mechanism. Recent studies show that the longitudinal muscle contraction (LMC)-related axial stretch might play a role in the LES relaxation by activating the mechanosensitive inhibitory motor neurons. The aim of our study was to determine whether the CCK-induced LES relaxation and the characteristics of LMC resemble those seen with spontaneous transient LES relaxation in humans. Nine healthy volunteers (5 Fr, 40 ± 12 yr) received escalating doses of CCK-octapeptide (CCK-8) (5, 10, 20, and 40 ng/kg). All subjects demonstrated a monophasic response to 5 ng/kg of CCK-8. In the majority of subjects, this response consisted of partial LES relaxation. All subjects showed a biphasic response to 40 ng/kg of CCK-8. The latter in most subjects consisted of 1) a period of partial relaxation followed by 2) complete LES relaxation along with crural diaphragm inhibition. The length of the esophagus decreased by 0.9 ± 0.4 cm, and muscle thickness increased by 40 ± 14% to 1.4 ± 0.2 mm (P < 0.05) during initial partial LES relaxation. During complete LES relaxation there was greater LMC, as demonstrated by an esophageal shortening of 1.9 ± 0.5 cm and an increase in muscle thickness of 100 ± 16% (P < 0.01). The complete phase 2 LES relaxation typically terminated with a robust after-contraction. Atropine significantly attenuated the CCK-induced esophageal LMC, prevented crural diaphragm inhibition, and abolished the phase 2 complete LES relaxation.

NEW & NOTEWORTHY The phenotypic features of CCK-induced longitudinal muscle contraction (LMC), complete lower esophageal sphincter (LES) relaxation, and crural diaphragm inhibition, followed by a robust after-contraction, resemble those seen during spontaneous transient LES relaxation. A strong temporal relationship between the LMC and complete transient LES relaxation supports our hypothesis that the LMC plays an important role in the LES relaxation and crural diaphragmatic inhibition.

Keywords: CCK, esophageal shortening, transient LES relaxation

INTRODUCTION

Cholecystokinin (CCK) is a peptide with two types of receptors (CCKA and CCKB), which are present throughout the central nervous system (36) and in the gastrointestinal tract (5). CCK receptor subtypes and their encoding gene are identical in the brain and gastrointestinal system (40). CCK administration in cats and healthy humans induces lower esophageal sphincter (LES) relaxation (4, 17, 35, 38). Studies in humans have shown that the CCKA receptor agonists increase the frequency of transient LES relaxation (TLESR), and CCKA receptor antagonists decrease the frequency of TLESR (7, 9, 10, 23, 39, 42). The TLESR is a dominant mechanism of gastroesophageal reflux in humans and has a distinct phenotype; e.g., 1) it is different from the swallow-induced LES relaxation in that the TLESR is significantly longer than the swallow-related relaxation, 2) it is associated with complete LES relaxation and crural diaphragm inhibition, and 3) it is associated with a strong contraction of the longitudinal muscle of the distal esophagus (2). Whether CCK-induced LES relaxation in humans is associated with all of the above features of spontaneous TLESR is not known.

Recent studies suggest that an axial stretch of the esophagus in the oral direction induces a neurally mediated LES relaxation through the direct activation of the inhibitory motor neurons of the myenteric plexus (20). Further studies show that the inhibitory motor neurons possess a stretch-sensitive/mechanosensitive mechanism and release nitric oxide when stretched (15). The significance of the above observations is that an axial stretch on the LES caused by longitudinal muscle contraction (LMC) may activate inhibitory motor neurons to induce LES relaxation (27). The LES moves in the oral direction, significantly more with TLESR than with a swallow (~4 cm vs. ~2 cm) because of the stronger LMC during TLESR (31). Whether CCK-induced LES relaxation, similar to the other types of LES relaxation, is associated with LMC of the esophagus is not known. The goal of our study was to investigate whether the phenotypic features and LMC pattern of the CCK-induced LES relaxation resemble those of a spontaneous TLESR.

METHODS

High-frequency intraluminal ultrasound (HFIUS) imaging records LMC of the esophagus. It is based on the principle that with the LMC, as the esophagus shortens in the axial direction, there is an increase in the thickness and cross-sectional area (CSA) of the muscularis propria layer (29, 30). To monitor both circular and longitudinal esophageal muscle activity, we recorded synchronized esophageal high-resolution pressure topography and HFIUS imaging of the esophagus in nine subjects using techniques described previously (1, 22).

Study population.

Nine healthy subjects (40 ± 12 yr; 4 men) with no history of gastrointestinal disease/surgery were enrolled in the study. The human investigation committee of the University of California, San Diego approved the study protocol, and each subject signed an informed consent before enrollment in the study.

Experimental protocol.

A catheter assembly consisting of a high-resolution manometry (HRM) catheter and an HFIUS catheter was prepared before each experiment. The two catheters were assembled in such a fashion that the ultrasound transducer was positioned between the two adjacent pressure sensors of the HRM catheter. The Manoscan 360-HRM catheter (Medtronic, Minneapolis, MN) contained 36 circumferential solid-state pressure sensors, spaced 1 cm apart. The HFIUS consisted of a 3.5-F, 20-MHz ultrasound catheter (CVIS, Sunnyvale, CA) that was interfaced to the HP Sonos 100 ultrasound imaging system. The time-encoded ultrasound images (Thalaner Electronics, Ann Arbor, MI), with an accuracy of one-hundredth of a second, were also recorded digitally on a DVD. Subjects fasted for 6 h before the recordings. Topical anesthetic agent was applied to the nose and throat of the subject, and the catheter assembly was positioned transnasally into the esophagus such that the ultrasound transducer was positioned oral to the distal esophageal cardiovascular compression (3). An intravenous line was established, and recordings were obtained in awake and quiet subjects while they lay in the recumbent position. Five micrograms of CCK-8 (Kinevac, Bracco Diagnostics) were mixed with 5 ml of sterile water to a concentration of 1 µg/ml. Escalating doses of CCK-8 (5, 10, 20, and 40 ng/kg) were injected intravenously, 5–10 min apart. After each dose of CCK-8, 10 ml of normal saline were used to flush the intravenous line, and we waited until the manometry tracing appeared to return to the baseline values for the next injection. Additionally, in five participants CCK-8 injection was repeated after intravenous administration of 1 mg of atropine sulfate (American Regent, Shirley, NY) to assess cholinergic contribution to the esophageal and LES motor response. The participant’s heart rate was monitored after atropine was administered, and when the heart rate increased by 20 beats/min (presumable anticholinergic effects), the highest dose of CCK-8 (40 ng/kg) injection was repeated.

Muscle thickness measurements.

Muscle CSA changes measured from the circumferential B-mode ultrasound images is an accurate marker of the LMC (21, 28, 30). However, measurements of the muscle CSA over prolonged periods are extremely time consuming (30). Several studies show an excellent correlation between the muscle CSA and muscle thickness as an indicator of the LMC (26, 30). Therefore, we decided to measure muscle thickness rather than muscle CSA as the marker of LMC. The synchronized ultrasound B-mode images embedded in the Manoview analysis software were digitized using a digital video recorder. We used ImageJ 1.51w software (National Institute of Health, Bethesda, MD) to determine the distance between the inner edge of circular muscle and outer edge of longitudinal muscle layer in millimeters in the B-mode ultrasound images, referred to as muscle thickness. Esophageal M-mode images were used to scout for changes in the esophageal muscle thickness over time and for display purposes.

Data analysis.

Esophageal pressure and HFIUS images were analyzed at baseline and after each dose of CCK-8. For each dose of administered CCK-8, the HRM was analyzed to determine the following: 1) the end-expiratory esophagogastric junction (EGJ) pressure (i.e., LES pressure); 2) the rhythmic inspiratory EGJ pressure augmentation (i.e., crural diaphragm contraction) during regular tidal respiration; 3) duration of esophageal contraction and distal contractile integral (DCI); and 4) the end-expiratory length of the esophagus (between the midpoint of upper esophageal sphincter to the midpoint of LES high-pressure segment, measured in centimeters). The crural diaphragm inhibition was defined when the inspiration-induced increase in the EGJ pressure was nearly equal to the gastric inspiratory pressure oscillation (2). Esophageal responses to CCK-8 were categorized as phase 1 or phase 2. Phase 1 was the initial or first phase, which started ~20–30 s after the CCK-8 injection and showed phasic esophageal body contractions associated with partial LES relaxation (crural diaphragm contractions preserved). Phase 1 usually lasted less than 90 s. Phase 2 was the delayed or second phase of the response, which occurred 60–90 s after the higher doses of CCK-8 injection (usually seen after phase 1) and was associated with complete transient LES relaxation and crural diaphragm inhibition. The ultrasound images were analyzed to determine the onset, duration, and amplitude of changes in the muscle thickness before and after the CCK-8 injection. The baseline muscle thickness was an average of 10 midexpiratory measurements during the time periods where no esophageal contraction was observed. Esophageal muscle thickness is reported in millimeters and also as a percentage of the baseline thickness.

Statistical methods.

Data are presented as means ± SD. Paired t-test, analysis of variance (ANOVA) with repeated measures (if needed), and post hoc Tukey’s test were used to determine the statistical significance, as deemed appropriate. P values were considered statistically significant if <0.05.

RESULTS

The baseline esophageal LES pressure and crural diaphragm inspiratory pressure augmentations were 27 ± 5 and 21 ± 6 mmHg, respectively. CCK-8 injections were administered 7.5 ± 3.2 min apart. Bolus injection of 5 ng/kg CCK-8 was typically not associated with any symptoms. The higher doses of CCK-8 (especially 40 ng/kg) induced symptoms of nausea and abdominal queasiness in all subjects. One participant reported heartburn after CCK-8 injection and two participants developed retching and emesis following the highest dose of CCK-8 injection (40 ng/kg). In five participants (who did not develop emesis after the CCK injections), atropine was administered (1 mg), followed by a repeat of CCK (40 ng/kg injection), 9.4 ± 0.9 and 11.8 ± 0.8 min, respectively, after the completion of serial CCK injections.

Esophageal pressure response.

All subjects showed the initial or phase 1 EGJ motor response, 26 ± 6 s following the CCK-8 injection that lasted for 48 ± 16 s. Phasic, nonperistaltic distal esophageal muscle contractions with a DCI of 796 ± 845 mmHg·cm·s were sometimes present during the phase 1 response. Lower doses of CCK-8 injection (5 and 10 ng/kg) resulted in partial LES relaxation (53% ± 17%) in seven subjects during phase 1 response (Fig. 1A). There was no inhibition of crural diaphragm, as measured by the inspiratory pressure augmentations (17 ± 6 mmHg, P > 0.5 compared with baseline). In the remaining two subjects, a mild LES contractile response was observed during the initial phase 1 response (Fig. 1B).

Fig. 1.

Fig. 1.

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Esophageal pressure topography (EPT) plots and superimposed esophagogastric junction (EGJ) e-sleeve pressure (orange tracing) show a monophasic EGJ response (either relaxation or contraction, phase 1) after lower dose of cholecystokinin (CCK) injection (5–10 ng/kg). A: example representing the majority of subjects, a few seconds after the CCK injection and saline flush (5 ng/kg), lower esophageal sphincter (LES) shows partial relaxation without crural diaphragm inhibition lasting for more than 60 s. B: in 2 of 9 subjects, a few seconds after the CCK injection (10 ng/kg) and saline flush, LES shows mild contractile response with inspiratory decrease in the EGJ pressure oscillations that lasted for 45 s.

With higher doses of CCK-8 (20 and 40 ng/kg), a delayed or phase 2 response was observed (Fig. 2A). The hallmark of the delayed response to CCK-8 injection was complete LES relaxation (88% ± 8%, Fig. 2B) and crural diaphragm inhibition (Fig. 2C), as detected by reduction in the inspiratory EGJ pressure augmentations (5 ± 3 mmHg, P < 0.001 compared with baseline). The onset of phase 2 response was 79 ± 38 s after the CCK-8 injection, typically after a period of partial LES relaxation (Fig. 3A), and lasted for 24 ± 6 s. Delayed phase 2 response usually terminated with a robust after-contraction (DCI = 6,192 ± 4,485 mmHg·cm·s). The phase 2 response was observed in all study subjects (Fig. 2C) with at least one of the CCK-8 injections, including two subjects who demonstrated the LES contractile response during phase 1 (Fig. 3B).

Fig. 2.

Fig. 2.

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Frequency and amplitude of esophagogastric junction (EGJ) phase 1 and phase 2 responses to cholecystokinin (CCK) injection. A: frequency of EGJ phase 1 and phase 2 relaxation response rates to escalating doses of CCK-octapeptide (CCK-8) [5, 10, 20, and 40 ng/kg (n = 9 subjects)]. B: EGJ relaxation during phase 2 of the CCK response is more complete (n = 7). Both end-expiratory and inspiratory augmentations of the EGJ pressure representing lower esophageal sphincter (LES) and crural diaphragm (CD) components are reduced significantly (ANOVA and post hoc Tukey’s test; *P < 0.05 compared with baseline and #P < 0.01 compared with baseline and phase 1). C: LES relaxation during phase 2 of the CCK response (n = 7, paired t-test; *P < 0.01 and #P < 0.001 compared with phase 1).

Fig. 3.

Fig. 3.

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Esophageal pressure topography (EPT) plots and superimposed esophagogastric junction (EGJ) e-sleeve pressures (orange tracing) show a biphasic EGJ response (both phase 1 and phase 2) after higher doses of cholecystokinin (CCK) injection (20–40 ng/kg). A: example representing the majority of subjects, a few seconds after CCK injection and saline flush (20 ng/kg), the LES shows initial partial relaxation without crural diaphragm inhibition that lasted for 60 s. This was followed by delayed complete transient LES relaxation and crural diaphragm inhibition that lasted for 20 s. B: in the same subject as in Fig. 1B with contractile phase 1 response, a few seconds after the higher dose of CCK injection (40 ng/kg) and saline flush, the LES showed a mild contractile response that lasted for 45 s. This was followed by the LES relaxation and crural diaphragm inhibition, which were terminated by a robust peristaltic esophageal contraction.

Esophageal longitudinal muscle response.

The esophageal LMC was assessed by two methods: 1) reduction in the axial length of the esophagus as measured by the HRM, and 2) changes in the muscle thickness recorded on the ultrasound images (Fig. 4, A and B). The baseline resting end-expiratory esophageal length and muscle thickness before CCK-8 injections were 25.7 ± 0.9 cm and 1.0 ± 0.2 mm, respectively. Irrespective of the relaxation or contractile nature of the LES response, esophageal muscle thickness increased by 40 ± 14% (1.4 ± 0.2 mm, P < 0.05) and esophageal length decreased by 0.9 ± 0.4 cm during phase 1 response (Fig. 5A). During phase 2 response, the esophageal length decreased by 1.9 ± 0.5 cm and muscle thickness increased by 100 ± 16% (Fig. 5B, P < 0.01). At the end of escalating doses of CCK-8 injection (32 ± 12 min) and during the recovery period, the LES pressure did not completely return to baseline and remained 13 ± 7 mmHg lower than the pre-CCK baseline. Furthermore, muscle thickness remained 20 ± 10% higher than the baseline muscle thickness (Fig. 4B, P < 0.05).

Fig. 4.

Fig. 4.

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Esophageal pressure topography (EPT) plots and superimposed esophagogastric junction (EGJ) e-sleeve pressures (orange tracing), along with synchronized intraluminal high-frequency B-mode (bottom panels) and M-mode (top panel) images showing typical biphasic EGJ responses (phase 1 and phase 2) associated with esophageal longitudinal muscle contraction. A: several seconds after the cholecystokinin (CCK) injection and saline flush (10 ng/kg), the lower esophageal sphincter (LES) shows a phase 1 response; i.e., partial LES relaxation without crural diaphragm inhibition, similar to a spontaneous swallow-induced LES relaxation. Note an increase in esophageal muscle thickness compared with the baseline. Forty-five seconds later, with the onset of phase 2 and diaphragm inhibition, the esophageal muscle thickness increased significantly (more than phase 1), as seen in both the ultrasound images and LES lift in the EPT plot. At the end of phase 2, a robust esophageal and LES after contraction led to the termination of “TLESR-like” phase 2 response. B: nearly 30 s after the CCK injection (40 ng/kg), the LES shows a phase 1 partial relaxation without crural diaphragm inhibition comparable to the preceding swallow-related LES relaxation. The increase in esophageal muscle thickness during this phase is smaller than during phase 2, as seen in the M-mode (top panel) image. Ninety seconds after the CCK, with the onset of phase 2 and diaphragm inhibition, note a marked increase in the esophageal muscle thickness (more than phase 1), as shown in both the ultrasound images and LES lift in the EPT plot. At the end of phase 2, a primary peristaltic contraction and LES after-contraction leads to the termination of the “TLESR-like” phase 2 response.

Fig. 5.

Fig. 5.

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Esophageal length and muscle thickness changes before and after cholecystokinin (CCK) injection (n = 9). A: baseline and during phase 1 partial response to CCK injections (ANOVA and post hoc Tukey’s test; *P < 0.05 and #P < 0.01 compared with the baseline).B: phase 2 complete response to CCK injections and during recovery period (ANOVA and post hoc Tukey’s test *P < 0.05 and #P < 0.01 compared with the recovery period).

Esophageal response to CCK-8 after atropine.

After atropine and before repeat CCK-8 injection, the resting end-expiratory esophageal length and muscle thickness were 25.8 ± 1.2 cm and 1.1 ± 0.1 mm, respectively (9% ± 3% reduction in muscle thickness, P < 0.05 compared with recovery period). Inspiratory crural diaphragm pressure augmentations were 17 ± 16 mmHg. After CCK, there was an increase in the muscle thickness (26 ± 7%, P < 0.05) to 1.4 ± 0.2 mm and esophageal shortening by 0.5 ± 1.1 cm (Fig. 6A, P > 0.1). Attenuated, phasic nonperistaltic esophageal contractions were occasionally observed (Fig. 6B). There were no discernable changes in the end-expiratory LES pressure (3 ± 4 mmHg, P > 0.3) and inspiratory pressure augmentations (9 ± 4 mmHg, P > 0.1). None of the five subjects displayed the phase 2 response; i.e., complete transient LES relaxation and crural diaphragm inhibition during the 5-min interval after the CCK injection.

Fig. 6.

Fig. 6.

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Esophageal pressure topography (EPT) plots and superimposed esophagogastric junction (EGJ) e-sleeve pressures (orange tracing) after atropine (1 mg) and cholecystokinin (CCK) injection (40 ng/kg). A: atropine increased the pulse rate by 20 beats/min and subsequent CCK injection no longer showed a discernable response on the EGJ pressure. The phase 2 complete relaxation was abolished by atropine. B: atropine increased pulse rate by 40 beats/min and subsequent CCK injection no longer showed a discernable response on the EGJ pressure despite the presence of attenuated phasic non-peristaltic esophageal contractions. The phase 2 complete relaxation was absent following atropine injection.

DISCUSSION

In summary, our data show that 1) CCK-8 induces a biphasic EGJ response, an initial partial LES relaxation without crural diaphragm inhibition followed by complete transient relaxation of LES, and crural diaphragm inhibition; 2) the changes in LES pressure are temporally and quantitatively associated with axial esophageal shortening, as measured by the changes in the esophageal length recorded by the HRM and LMC recorded by the ultrasound images; and 3) atropine attenuates the CCK-induced LMC, prevents crural diaphragm inhibition, and abolishes the complete transient LES relaxation phase 2 response. Overall, the phenotypic appearance of the late, or phase 2, response resembles that of a spontaneous TLESR; that is, it is prolonged in duration and associated with complete relaxation of the LES and crural diaphragm, strong LMC of the distal esophagus, and typical termination of the LES relaxation by a robust after-contraction in the esophagus.

The inhibitory effect of CCK-8 on the LES pressure in humans has been known since the 1970s (17, 35, 38). The studies in cats demonstrate that CCK-8 induces a neurally mediated LES relaxation through the CCKA receptor and myogenic contraction through the CCKB receptor located on the LES smooth muscles (4, 32). Rattan studied the structure-activity relationship of the subtypes of CCK receptors on the cat LES and came to the conclusion that the CCK A receptor located on the myenteric neurons is critically dependent on sulfation (SO4) and induced LES relaxation (32). On the other hand, the CCKb receptors located on the LES smooth muscle cause LES contraction and do not require SO4 (15). Dodds et al. found that unlike normal subjects, in whom the CCK induces LES relaxation, CCK causes LES contraction in patients with achalasia esophagus. The latter was explained based on the absence/degeneration of the inhibitory neurons in achalasia esophagus. The authors proposed that the CCK may be used as a test of the denervation of the esophagus (13).

The interest in the role of CCK resurfaced with the observation that intravenous infusion of CCK-8 increases the gastric distension-induced TLESR frequency in dogs (8) and CCKa receptor antagonist but not the CCKb receptor antagonist, which decreases the TLESR frequency (9, 23). Other studies found the same in humans (7, 10, 39, 42). All the above observations were conducted with continuous intravenous infusion of CCK agonists and antagonists. The focus of these studies was to determine the effects of CCK on the frequency of TLESR rather than on the acute effect of CCK bolus injection on the LES and crural diaphragm response. The novel and unique aspect of our study is that we studied the acute effects of bolus intravenous injection of CCK on the two components of the sphincter mechanism at the EGJ using the pressure recording techniques (HRM), which, in addition to measuring LES pressure, can also reveal axial shortening of the esophagus. We also measured LMC using the ultrasound imaging method and found a close temporal and quantitative relationship between the LMC and LES relaxation. Studies have shown that a strong LMC is an important component of the spontaneous TLESR (2, 31). In fact, the phenotypic response seen during phase 2, following CCK injection, precisely resembles that of a spontaneous TLESR.

The major impetus to conduct this study was to determine whether the CCK-induced LES relaxation was accompanied by LMC because of recent observations related to the role of axial stretch in inducing LES relaxation. These studies from our laboratory show that an axial stretch on the esophagus in oral direction induces a neurally mediated LES relaxation (14), an effect mediated by the activation of inhibitory motor neurons in response to mechanical stretch (20). These neurons are mechanosensitive and release nitric oxide in response to mechanical stretch (15). The above observations have implications as to the mechanism of swallow-induced LES relaxation and TLESR. The traditional thinking is that a swallow activates vagal efferent nerves that synapse with the inhibitory motor neurons to induce LES relaxation. It is possible that the vagus nerve actually activates LMC, which in turn activates inhibitory motor neurons through a stretch-sensitive or mechanosensitive mechanism to induce LES relaxation (27). The findings in the current study of temporal and quantitative correlation between LMC and LES relaxation support that the CCK-induced LES relaxation may be also mediated by the stretch-sensitive mechanism of the inhibitory motor neurons activated by the LMC. There is also a close correlation between an axial stretch in the oral direction and crural diaphragm inhibition (25) through a peripheral mechanism. Our study shows that atropine inhibits CCK-induced LMC and crural diaphragm inhibition. The findings support the role of axial stretch mediated crural diaphragm inhibition. One can argue that the close association between LMC and LES relaxation, along with crural diaphragm inhibition, in our current human study does not prove causality, which might be considered a limitation of our study. Obviously, the experiments required to prove causality cannot be performed on the human subjects. On the other hand, if our contention that the CCK-induced LES relaxation is caused by LMC is true, one may speculate that the CCK-8 working through CCKA receptor only causes muscle contraction and the resulting LMC activates stretch-sensitive inhibitory motor neurons to induce LES relaxation. In fact, careful studies by Gonzalez et al. showed that, in humans, the inhibitory effect of CCK on the LES is mediated through an extra-sphincteric CCKA receptor, and even the excitatory effects of CCK are probably mediated through the CCKA receptors (18).

In 2 of the 9 subjects in our study, we found LES contraction instead of relaxation during phase 1 of the CCK response. In the remainder, only the LES relaxation was observed. The reason for the difference is not clear. The CCK-8 induced LES contraction instead of LES relaxation in opossums, suggesting species differences (11). Behar et al. and Dodds et al. also observed LES contractile response in 4 of 51 cats (4) and 1 of 7 human subjects, respectively (13). In our two cases, swallow-induced LES relaxation was present and phase 2 responses with “TLESR-like” phenotype (complete LES relaxation and crural diaphragm inhibition) were also present, arguing against subclinical esophageal denervation. There was axial shortening of the esophagus, and LMC contraction was present during the LES contraction, which would contradict our hypothesis of LMC and LES relaxation. We can only speculate that there might be heterogeneity in the CCK receptor-mediated esophageal motor responses among individuals, just like there are species differences in the effects of CCK on the LES of cats and opossums.

Our study is unable to determine if the primary action of CCK-8 is on the gastric tone and gastric mechanoreceptors. Previous studies using a barostat have shown that the exogenous CCK infusion or endogenous CCK surge reduce the gastric fundus tone in humans (37, 42). Single fiber recordings from the vagal afferents found that the CCK causes gastric relaxation and suppress discharge from the distention responsive gastric mechanoreceptors in the rat (19). We cannot exclude the possibility that the relaxation-induced gastric distension may trigger transient LES relaxation. The higher doses of CCK-8 we used may be considered supraphysiological because the serum levels exceed expected postprandial peak plasma levels (24, 34). This would be especially true if the effects of exogenous CCK-8 were entirely hormonal. The CCK receptors are known to be abundantly present in the central and peripheral nervous system, where CCK-8 is the predominant molecular form and may act as a neurotransmitter (12, 41). The dominant isoforms of CCK in the plasma are, however, larger [CCK-58 and CCK-39 (16), CCK-33 and CCK-22 (24, 34)], especially during postprandial peak activity. As shown previously, circulating CCK-8 may activate central neuronal CCK receptors as a neurotransmitter (33) or act in a paracrine fashion, as happens with mucosal vagal afferents (6). We suspect that the observed actions of CCK-8 may indicate neuronal or paracrine functions, rather than the hormonal function.

The interest on the effects of CCK on LES has been there for a long time and for many reasons, which include the following: 1) CCK, along with gastrin, may be involved in the neurohumoral control of LES tone; 2) CCK may be involved in the vagal inhibitory pathway to the LES; 3) CCK may be used as a diagnostic test of the denervation of the esophagus; and 4) CCKA receptors may be a desirable therapeutic target to reduce the frequency of TLESR to treat gastroesophageal reflux disease. Our interest to study the effects of CCK on the LES was different from all of the above and purely a scientific curiosity as to whether the effects of CCK on the LES function are mediated through the LMC. In this human study, we could only show association between the LMC and “TLESR-like” phenotype induced by bolus intravenous injection of CCK. Our study does not prove causality for which detailed pharmacological experimentation in the animal is required and can be the subject of future studies.

GRANTS

This publication was supported in part by National Institutes of Health Grant R01DK109376 (to R. Mittal) and the National Center for Research Resources and National Center for Advancing Translational Sciences Grant 8UL1TR000055.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.B. and R.K.M. conceived and designed research; A.B. performed experiments; A.B. analyzed data; A.B. and R.K.M. interpreted results of experiments; A.B. prepared figures; A.B. drafted manuscript; A.B. and R.K.M. edited and revised manuscript; A.B. and R.K.M. approved final version of manuscript.

ACKNOWLEDGMENTS

Present address of A. Babaei: Division of Gastroenterology, Dept. of Medicine, National Jewish Health, 1400 Jackson St., Denver, CO 80206 (e-mail: babaeia@njhealth.org).

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