Extracellular Calcium-Sensing Receptor Mediates Different Patterns of Intracellular Calcium Oscillations in Colonic Epithelial Cells

OSVALDO REY; STEVEN H YOUNG; ENRIQUE ROZENGURT

Congreso; Digestive Diseases Week; 2010

AGA

Background: The extracellular Ca2+-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However the signaling mechan- isms involved remain poorly defined. Here, we examined signaling pathways activated by the CaR, including Ca2+ oscillations, in individual colonic epithelial cells. Results: Single cell imaging of human-colon derived epithelial cells expressing the CaR, including SW-480, HT-29 and Caco-2 cells, shows that stimulation of this receptor by addition of aromatic amino acids, including L-phenylalanine, or by an elevation of the extracellular Ca2+ concentration (5 mM) promoted striking intracellular Ca2+ oscillations. Analysis of SW-480 cells showed that the intracellular Ca2+ oscillations in response to extracellular Ca2+ were of sinusoidal pattern (mean frequency of 3.5 +/- 0.8 min-1), whereas those induced by 5 mM L-phenylalanine were of transient type, i. e. Ca2+ spikes that returned to baseline levels (mean frequency of 1.5 +/- 0.2 min-1). Furthermore, extracellular Ca2+-mediated stimulation of the CaR elicits sinusoidal intracellular Ca2+ oscillations through the PLC/DAG/Ins (1,4,5)P3 pathway, as shown by a biosensor that detects the accumulation of DAG in the plasma membrane. We generated CaR mutants, including E127A, S165A, L174A, R185Q, D190A, W206A, Y218A, N287A, E297A, W299A and G319A that further separate the signaling pathways initiated by stimulation of the CaR by external Ca2+ and aromatic amino acids in colonic epithelial cells. Conclusion: It is increasingly recognized that the pattern and frequency of intracellular Ca2+ oscillations encodes information that differentially regulates fundamental biological responses, including signal transduction, metabolism, selective gene expression and cell proliferation. Our results demonstrate, for the first time, that the CaR triggers different patterns of intracellular Ca2+ oscillations in colonic epithelial cells.