Abstract
Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.
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