Flow injection microscopy for the study of intracellular calcium mobilization by muscarinic agonists

The study of cellular response to chemical agonists is essential in understanding the complex functions mediated by cell surface receptors. Flow injection microscopy has been used with the CHO-M1-WT3 cell line and the fluorescent Ca2+ indicator Fura-2-AM to monitor mobilization of internal Ca2+, Repeated stimulation of cells mounted in an inverted radial flow chamber allows the direct comparison of relative intracellular Ca2+ mobilization with respect to agonist dose. The process of determining dose-response relationships is simplified since an entire dose-response curve can be constructed from a distinct set of cells. Use of flow injection lends precision to the application and removal of agonists while allowing cellular activity to be monitored throughout the stimulation and recovery processes. In this work, dose-response curves have been constructed for the muscarinic agonists carbachol, acetylcholine, and pilocarpine resulting in EC50 values of 1.7 mu M, 56 nM, and 6.8 mu M, respectively. (C) 1999 Academic Press.