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 th e expression of ryanodine receptor (RyR) isoforms in gut epithelial ce lls. Restriction digest and sequence analysis of the PCR product showe d the presence of RyR 2 and RyR 3. [H-3]Ry binding studies on a micros ome preparation, in a high-salt buffer, showed specific binding with a n EC(50) of 15 mu M. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to a cetylcholine. At ail concentrations used acetylcholine induced sinusoi dal cytosolic Ca2+ concentration ([Ca2+](i)) oscillations. In response to 10(-4) M acetylcholine, levels of inositol 1,4,5-trisphosphate (In sP(3)) showed a peak of six times the basal level, at 30 s after stimu lation. Application of caffeine alone failed to elicit a rise in cytos olic Ca2+. However, caffeine (5-50 mM) did rapidly and reversibly inhi bit the acetylcholine-induced [Ca2+](i) oscillations. The effects of R y were more complex. Applied alone, Ry had no effect on the [Ca2+](i) signal. When applied during agonist-evoked [Ca2+](i) oscil lations, Ry (10 mu M) slowly blocked the response. In the continuous presence of Ry (10 mu M) a short application of acetylcholine elicited a [Ca2+](i) response that continued as oscillations even when the agonist was rem oved. The oscillations, in the presence of Ry (10 mu M) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by a n application of a higher concentration of Ry (100 mu M). These effect s 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, identifie d by RT-PCR, play a central role in [Ca2+](i) oscillations in gut epit helial cells.