Identification and function of type-2 and type-3 ryanodine receptors in gut epithelial cells

Reverse transcription-PCR (RT-PCR) techniques were used to identify the expression of ryanodine receptor (RyR) isoforms in gut epithelial cells. Restriction digest and sequence analysis of the PCR product showed the presence of RyR 2 and RyR 3. [3H]Ry binding studies on a microsome preparation, in a high-salt buffer, showed specific binding with an EC50 of 15 µM. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to acetylcholine. At all concentrations used acetylcholine induced sinusoidal cytosolic Ca2+ concentration ([Ca2+]i) oscillations. In response to 10-4 M acetylcholine, levels of inositol 1,4,5-trisphosphate (InsP3) showed a peak of six times the basal level, at 30 s after stimulation. Application of caffeine alone failed to elicit a rise in cytosolic Ca2+. However, caffeine (5–50 mM) did rapidly and reversibly inhibit the acetylcholine-induced [Ca2+]i oscillations. The effects of Ry were more complex. Applied alone, Ry had no effect on the [Ca2+]i signal. When applied during agonist-evoked [Ca2+]i oscillations, Ry (10 µM) slowly blocked the response. In the continuous presence of Ry (10 µM) a short application of acetylcholine elicited a [Ca2+]i response that continued as oscillations even when the agonist was removed. The oscillations, in the presence of Ry (10 µM) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by an application of a higher concentration of Ry (100 µM). These effects are consistent with the known use-dependence and dose-dependence for Ry action at the RyR. We conclude that the RyR 2 and RyR 3, identified by RT-PCR, play a central role in [Ca2+]i oscillations in gut epithelial cells