Establishing A Cell Model Of Calcium Imaging For Bitterness Evaluation

Human have five basic tastes, including sweet, salty, bitter, sour and umami. Bitter taste is one of the most stimulating ones and has an important role in the development of oral pharmaceuticals. With respect to patient acceptability and compliance, bitter taste is one of the major factors determining the commercial success of oral formulations, especially in pediatric medicine. Hence, it has become a hot issue for pharmaceutical industries to develop palatable and pleasant-tasting products. The industry adopts various kinds of taste-masking techniques to develop an appropriate formulation. Taste assessment is becoming more significant as an important quality-control parameter for evaluation of taste-masked formulations.With the development of taste-masking technologies, the taste assessment has also made great progress. In both in vivo and in vitro studies, such as human taste panel studies, electrophysiological methods, animal preference studies and the electronic tongue, the taste assessment has experienced great transformation from subjective to objective, from qualitative to quantitative and from low detecting efficiency to high-throughput. To date, human taste panel test is still most commonly used. Although such assessment is traditional and intuitive, it is constrained by the difference among the individual volunteers and the risks from uncertainties with the security of drugs. Electronic tongue is the most promising method in bitter taste evaluation. It has many advantages, such as objectivity, good reproducibility and fast detection and so on. However, it depends largely on the detecting sensor and is difficult to maintain.We sought to build a cell platform for evaluating bitter levels in a high-throughput way. To date,25 human bitter taste receptors (TAS2R) have been identified, which all belong to the seven-transmembrane G-protein-coupled receptor (GPCR) family. As suggested by the bitter taste transduction mechanism, when bitter substance and the receptor protein interact, G-proteins that act cooperatively with bitter receptors are activated, which in turn activates the cellular signaling pathways that depend on the proteins of Ga, Gp and Gy. As a result, the level of intracellular calcium is elevated. To simulate such mechanism, a bitter evaluating platform is built by cell transfection. Human 46th bitter taste receptor is chosen and is conjugated with bovine rhodopsin membrane localization sequence within a plasmid. The hT2R46-BR is co-expressed with a plasmid expressing Ga in HEK293. Then the transfected cells are stimulated using different bitter substances. Measurement of real-time changes in intracellular calcium concentration by calcium imaging techniques will allow evaluation of bitter levels. However, due to the limitations from the transfection, the constructed bitterness evaluation platform suffered from inadequate efficiency. So we also built a platform that can evaluate bitterness by incorporating taste receptors on smooth muscle cells isolated from animal’s airways. In this article, preliminary validation has been done by measuring calcium levels with real-time calcium imaging in the cell level.