Gastroenterology

Gastroenterology

Volume 138, Issue 4, April 2010, Pages 1479-1490
Gastroenterology

Basic—Alimentary Tract
Cocaine- and Amphetamine-Regulated Transcript Mediates the Actions of Cholecystokinin on Rat Vagal Afferent Neurons

https://doi.org/10.1053/j.gastro.2009.10.034Get rights and content

Background & Aims

Cholecystokinin (CCK) acts on vagal afferent neurons to inhibit food intake and gastric emptying; it also increases expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART), but the significance of this is unknown. We investigated the role of CARTp in vagal afferent neurons.

Methods

Release of CART peptide (CARTp) from cultured vagal afferent neurons was determined by enzyme-linked immunosorbent assay. Expression of receptors and neuropeptides in rat vagal afferent neurons in response to CARTp was studied using immunohistochemistry and luciferase promoter reporter constructs. Effects of CARTp and CCK were studied on food intake.

Results

CCK stimulated CARTp release from cultured nodose neurons. CARTp replicated the effect of CCK in stimulating expression of Y2R and of CART itself in these neurons in vivo and in vitro, but not in inhibiting cannabinoid-1, melanin-concentrating hormone, and melanin-concentrating hormone-1 receptor expression. Effects of CCK on Y2R and CART expression were reduced by CART small interfering RNA or brefeldin A. Exposure of rats to CARTp increased the inhibitory action of CCK on food intake after short-, but not long-duration, fasting.

Conclusions

The actions of CCK in stimulating CART and Y2R expression in vagal afferent neurons and in inhibiting food intake are augmented by CARTp; CARTp is released by CCK from these neurons, indicating that it acts as an autocrine excitatory mediator.

Section snippets

Materials

CCK8s and ghrelin were obtained from Bachem (St. Helens, Merseyside, UK); leptin and phorbol 12-acetate-13-myristate ester (PMA) were obtained from Sigma (Poole, Dorset, UK). CARTp (55–102, human) was obtained from American Peptide Company (Sunnyvale, CA). Brefeldin-A (BFA) was obtained from Epicentre Biotechnologies (Madison, WI).

Animals

Adult male Wistar rats (225–300 g) were housed at 22°C under a 12-hour light/dark cycle with ad libitum access to food and water, unless stated otherwise. Studies

CCK Releases CARTp From Nodose Ganglion Neurons

To determine whether CARTp is secreted from vagal afferent neurons in response to stimulation, we cultured neurons for 72 hours and then transferred cells into serum-free medium overnight before treatment with CCK8s for 2 hours. The concentration of CARTp in media from unstimulated cells was 113 ± 20 pg/mL and in response to CCK8s was increased approximately 3-fold (P < .001). Leptin augmented the action of CCK8s and ghrelin inhibited it (P < .001 in both cases), but neither had any action

Discussion

Vagal afferent neurons serving the gut mediate the effects of a variety of peripheral signals on food intake and gastric emptying. One of the best studied hormones stimulating these neurons is CCK, and previous studies have shown that this is associated with inhibition of gastric emptying,5, 6 inhibition of food intake,8, 34 stimulation of pancreatic secretion,35 and inhibition of gastrointestinal inflammatory responses.36 We have reported that CCK stimulates expression in vagal afferent

Acknowledgments

We are grateful to Drs M. Kuhar and D. Ginty for the gift of plasmids.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by grants from the Biotechnology and Biological Sciences Research Council and MRC (GJD), and National Institutes of Health (HER, NIHDK41004).

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