Abstract
Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction. Here, we introduce the methods to isolate the mouse ileal crypts, which consist of various types of cells including EC cells, and to analyze the intracellular calcium dynamics. Real tissue was inserted with a plastic straw and the smooth muscle layers were peeled off. The mucosa were digested with collagenase and then suspended by moderate pipetting. Ileal crypts were separated by stepwise filtrations through 2 different nylon-meshes. The isolated crypt contained 0-3 EC cells as identified by immunostaining using anti-5-HT antibody followed by confocal microscopy. Isolated crypts were attached to a coverglass by an adhesive material (Cell-Tak) and loaded with fura-2/AM. Intracellular calcium dynamics in EC cells were obtained by digital video-imaging analysis of fura-2 fluorescence followed by the identification of EC cells with immunostaining of 5-HT granules. By these methods, it was suggested that norepinephrine elicited a transient elevation of intracellular calcium concentration in EC cells via α2-adrenoceptors. These methods could be also useful to analyze the signal transduction system in intestinal endocrine cells that contain various intestinal hormones such as gastrin, cholecystokinin or secretin.
