Background & aims: Acetylcholine-induced calcium signaling dynamics have been described in cell monolayers derived from colonic mucosa, but not in intact colonic crypts. The aim of this study was to characterize the spatiotemporal characteristics of calcium signaling induced by acetylcholine in isolated intact rat colonic crypts and to identify the muscarinic receptor subtype coupled to this signaling pathway.
Methods: Isolated crypts from the distal colon of male Wistar rats were loaded with the calcium-sensitive dye Fura-2 and imaged with a charge-coupled device video camera.
Results: Acetylcholine mobilized intracellular calcium with an EC50 of 3.9 micromol/L. The response was initiated at the base of the crypt and progressed toward the surface. The velocity of propagation was dose dependent. Addition of muscarinic antagonists inhibited the response (pKb values calculated for pirenzepine and 4-DAMP, 6.08 and 8.65, respectively). Microperfusion of acetylcholine initiated a calcium signal throughout the lower half of the crypt. Microinjection of inositol 1,4,5-triphosphate induced a propagation of a calcium signal along the crypt axis. Heptanol inhibited the velocity of acetylcholine-induced wave propagation by 33%.
Conclusions: M3 muscarinic receptors are coupled to the mobilization of calcium from intracellular stores of intact, isolated rat colonic crypts. Intercellular communication potentiates the propagation of the acetylcholine-induced calcium signal along the crypt axis.