Amino acids stimulate cholecystokinin release through the Ca²⁺-sensing receptor

Abstract

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca2+-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca2+ but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca2+-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca2+ ([Ca2+]i). The increase in [Ca2+]i was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K+ channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.

29 Immunoglobulin-Like Domain Containing Receptor Mediates Fat-Stimulated Cholecystokinin Secretion

“…Several physiological secretagogues (such as monitor peptide and amino acids) as well as exposure to depolarizing concentrations of KCl increase [Ca 2+ ] i , which is linked to hormone release (7,8,23,24). In this study, involvement of calcium signaling in ILDR1-stimulated CCK secretion was demonstrated by comparing [Ca 2+ ] i in wild-type and Ildr1 -/-CCK-EGFP cells.…”

“…Like most gastrointestinal hormones, CCK is released into the blood following ingestion of a meal, and despite the apical surface being exposed to the lumen of the intestine, the mechanisms by which foods stimulate hormone secretion are largely unknown. It has recently been demonstrated that CCK cells contain calcium-sensing receptors that mediate amino acid-stimulated CCK release (7,8), supporting the concept that cell surface receptors regulate food-stimulated gastrointestinal hormone release. Of the food components that stimulate CCK secretion, fats are the most important.…”

“…Intestinal cells were dissociated and subjected to FACS as described previously (8). RNA was isolated using the RNeasy Micro Kit (Qiagen) following manufacturer's recommendations.…”

“…Lipoproteins (100 μg protein/ml final concentration) in the presence or absence of C12 (100 μM final, 20 mM stock prepared in isopropanol) were added to the wells, and the plate was incubated for 30 minutes at 37°C in a water bath. After 30 minutes, cells were chilled on ice for 5 minutes and centrifuged at 1,500 g (average g) for 5 minutes at 4°C, and the supernatant was carefully removed for measurement of CCK release by radioimmunoassay (8). Assays were performed in triplicate or quadruplicate, and data were normalized to basal release.…”

“…Calcium signaling in CCK cells has been shown to be critically important for regulated hormone secretion (8,25,26). Several physiological secretagogues (such as monitor peptide and amino acids) as well as exposure to depolarizing concentrations of KCl increase [Ca 2+ ] i , which is linked to hormone release (7,8,23,24).…”