A new role for the extracellular calcium-sensing receptor demonstrated by using CCK-eGFP BAC mice

Abstract

cesses both exciting and fun is the application of new techniques to address a problem where understanding has been constrained by previous techniques. Two complementary papers (10, 19) in this issue illustrate such an application. The new technique is the use of bacterial artificial chromosome (BAC) transgenic mice. The problem is nutrient sensing by enteroendocrine cells. Widely used in neuroscience research (5), the BAC mice allow either targeted gene deletion or overexpression (20). Many transgenic BAC mice express regulatory sequences together with enhanced green fluorescent protein (eGFP), which also allows identification of specific cell types. The eGFP allows these cells to be recovered using FACS for further analysis. Such BAC transgenic mice may then be mated with knockout or reporter mice to further define the presence of either a receptor or a pathway activated by the receptor. The satiating effects of dietary protein may be due in part to cholecystokinin (CCK) secretion from duodenal I cells. Although aromatic amino acids have been understood to be mediators of CCK secretion from these cells, the technical difficulties of recovering sufficient amounts of homogeneous I cells from fetal or adult intestine are character building. The use of cell lines (i.e., STC-1) that are tumorigenic but endocrine derived is informative but equivocal. Consequently, little is understood about the cellular mechanism by which amino acids affect CCK secretion. The extracellular calcium-sensing receptor (CaSR), originally cloned from the parathyroid, but found in distinct regions of the gastrointestinal system, may be activated by aromatic amino acids, consistent with the CaSR being an amino acid sensor (1, 2). Because the CaSR knockout is perinatal lethal, it was “rescued” by taking parathyroid hormone (PTH) out of the system through breeding with CaSR / PTH / heterozygotes. The resulting CaSR / PTH / are sustained with 2% Ca 2 in their drinking water (8, 9). Analyses using heterologous CaSR expressing cells have shown differences in intracellular Ca 2 oscillation frequency (13, 14) generated by aromatic amino acids compared with other ligands, but direct in vivo evidence from enteroendocrine cells has been lacking. Indeed, we still don’t understand the physiological importance of circulating amino acids in regulating the CaSR’s activity outside the gastrointestinal tract (3). The study by Liou et al. (10) from the laboratories of Raybould and Wank is as impressive as it is elegant. They used FACS to isolate and enrich duodenal I cells from the CCKeGFP BAC transgenic mice and demonstrated that the CaSR